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Sample records for secretome reveals proteins

  1. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

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    Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

    2011-11-01

    Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive

  2. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

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    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  3. Secretomes of Mycoplasma hyopneumoniae and Mycoplasma flocculare reveal differences associated to pathogenesis.

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    Paes, Jéssica A; Lorenzatto, Karina R; de Moraes, Sofia N; Moura, Hercules; Barr, John R; Ferreira, Henrique B

    2017-02-10

    Mycoplasma hyopneumoniae and Mycoplasma flocculare cohabit the porcine respiratory tract. However, M. hyopneumoniae causes the porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. Comparative analyses demonstrated high similarity between these species, which includes the sharing of all predicted virulence factors. Nevertheless, studies related to soluble secretomes of mycoplasmas were little known, although they are important for bacterial-host interactions. The aim of this study was to perform a comparative analysis between the soluble secreted proteins repertoires of the pathogenic Mycoplasma hyopneumoniae and its closely related commensal Mycoplasma flocculare. For that, bacteria were cultured in medium with reduced serum concentration and secreted proteins were identified by a LC-MS/MS proteomics approach. Altogether, 62 and 26 proteins were identified as secreted by M. hyopneumoniae and M. flocculare, respectively, being just seven proteins shared between these bacteria. In M. hyopneumoniae secretome, 15 proteins described as virulence factors were found; while four putative virulence factors were identified in M. flocculare secretome. For the first time, clear differences related to virulence were found between these species, helping to elucidate the pathogenic nature of M. hyopneumoniae to swine hosts. For the first time, the secretomes of two porcine respiratory mycoplasmas, namely the pathogenic M. hyopneumoniae and the commensal M. flocculare were compared. The presented results revealed previously unknown differences between these two genetically related species, some of which are associated to the M. hyopneumoniae ability to cause porcine enzootic pneumonia. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

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    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  5. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

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    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  6. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

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    Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

    2010-08-06

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

  7. Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.

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    Bagordakis, Elizabete; Sawazaki-Calone, Iris; Macedo, Carolina Carneiro Soares; Carnielli, Carolina M; de Oliveira, Carine Ervolino; Rodrigues, Priscila Campioni; Rangel, Ana Lucia C A; Dos Santos, Jean Nunes; Risteli, Juha; Graner, Edgard; Salo, Tuula; Paes Leme, Adriana Franco; Coletta, Ricardo D

    2016-07-01

    An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-β1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

  8. Creation of a Human Secretome: A Novel Composite Library of Human Secreted Proteins: Validation Using Ovarian Cancer Gene Expression Data and a Virtual Secretome Array.

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    Vathipadiekal, Vinod; Wang, Victoria; Wei, Wei; Waldron, Levi; Drapkin, Ronny; Gillette, Michael; Skates, Steven; Birrer, Michael

    2015-11-01

    To generate a comprehensive "Secretome" of proteins potentially found in the blood and derive a virtual Affymetrix array. To validate the utility of this database for the discovery of novel serum-based biomarkers using ovarian cancer transcriptomic data. The secretome was constructed by aggregating the data from databases of known secreted proteins, transmembrane or membrane proteins, signal peptides, G-protein coupled receptors, or proteins existing in the extracellular region, and the virtual array was generated by mapping them to Affymetrix probeset identifiers. Whole-genome microarray data from ovarian cancer, normal ovarian surface epithelium, and fallopian tube epithelium were used to identify transcripts upregulated in ovarian cancer. We established the secretome from eight public databases and a virtual array consisting of 16,521 Affymetrix U133 Plus 2.0 probesets. Using ovarian cancer transcriptomic data, we identified candidate blood-based biomarkers for ovarian cancer and performed bioinformatic validation by demonstrating rediscovery of known biomarkers including CA125 and HE4. Two novel top biomarkers (FGF18 and GPR172A) were validated in serum samples from an independent patient cohort. We present the secretome, comprising the most comprehensive resource available for protein products that are potentially found in the blood. The associated virtual array can be used to translate gene-expression data into cancer biomarker discovery. A list of blood-based biomarkers for ovarian cancer detection is reported and includes CA125 and HE4. FGF18 and GPR172A were identified and validated by ELISA as being differentially expressed in the serum of ovarian cancer patients compared with controls. ©2015 American Association for Cancer Research.

  9. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

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    Whittington Jessica

    2007-07-01

    Full Text Available Abstract Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2- during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites, lipoprotein signal peptides (13 have SpII sites, and N-terminal membrane helices (9 have transmembrane helices. The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa of protective antigen (PA were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and

  10. Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

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    Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.; Paša-Tolić, Ljiljana; Chaput, Dominique L.; Wu, Si; Santelli, Cara M.; Hansel, Colleen M.

    2017-09-01

    Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and custom bioinformatic analyses to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that although the fungi produce a similar suite of extracellular enzymes, they exhibit striking differences in the regulation of these enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Paraconiothyrium sporulosum AP3s5-JAC2a and Alternaria alternata SRC1lrK2f employ sequential enzyme secretion patterns concomitant with decreasing resource availability, Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among cellulose-degrading Ascomycetes and enhances our understanding of their role in C turnover in the environment.

  11. A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures

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    Lee, Jee Yong; Kim, Hyun Ji; Yi, Jae Youn

    2016-01-01

    A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allows communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation-induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-(PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. Control of inflammatory waves, improved wound healing, and stabilization of the skin barrier are imperative for minimizing such injuries. Therefore, PPARα agonists and antagonists have the potential to become important therapeutic agents for the treatment of γ-irradiation induced skin damage. Specifically, our analysis suggests that the undesirable consequences of long-term exposure to ionizing radiation could be alleviated by PPARα agonists

  12. A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures

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    Lee, Jee Yong; Kim, Hyun Ji; Yi, Jae Youn [Korea Institute of Radiation and Medical Sciences, Daejeon (Korea, Republic of)

    2016-05-15

    A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allows communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation-induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-(PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. Control of inflammatory waves, improved wound healing, and stabilization of the skin barrier are imperative for minimizing such injuries. Therefore, PPARα agonists and antagonists have the potential to become important therapeutic agents for the treatment of γ-irradiation induced skin damage. Specifically, our analysis suggests that the undesirable consequences of long-term exposure to ionizing radiation could be alleviated by PPARα agonists.

  13. Defining the predicted protein secretome of the fungal wheat leaf pathogen Mycosphaerella graminicola.

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    Alexandre Morais do Amaral

    Full Text Available The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328 which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks, and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.

  14. Secretome weaponries of Cochliobolus lunatus interacting with potato leaf at different temperature regimes reveal a CL[xxxx]LHM - motif.

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    Louis, Bengyella; Waikhom, Sayanika Devi; Roy, Pranab; Bhardwaj, Pardeep Kumar; Singh, Mohendro Wakambam; Goyari, Sailendra; Sharma, Chandradev K; Talukdar, Narayan Chandra

    2014-03-20

    Plant and animal pathogenic fungus Cochliobolus lunatus cause great economic damages worldwide every year. C. lunatus displays an increased temperature dependent-virulence to a wide range of hosts. Nonetheless, this phenomenon is poorly understood due to lack of insights on the coordinated secretome weaponries produced by C. lunatus under heat-stress conditions on putative hosts. To understand the mechanism better, we dissected the secretome of C. lunatus interacting with potato (Solanum tuberosum L.) leaf at different temperature regimes. C. lunatus produced melanized colonizing hyphae in and on potato leaf, finely modulated the ambient pH as a function of temperature and secreted diverse set of proteins. Using two dimensional gel electrophoresis (2-D) and mass spectrometry (MS) technology, we observed discrete secretomes at 20°C, 28°C and 38°C. A total of 21 differentially expressed peptide spots and 10 unique peptide spots (that did not align on the gels) matched with 28 unique protein models predicted from C. lunatus m118 v.2 genome peptides. Furthermore, C. lunatus secreted peptides via classical and non-classical pathways related to virulence, proteolysis, nucleic acid metabolism, carbohydrate metabolism, heat stress, signal trafficking and some with unidentified catalytic domains. We have identified a set of 5 soluble candidate effectors of unknown function from C. lunatus secretome weaponries against potato crop at different temperature regimes. Our findings demonstrate that C. lunatus has a repertoire of signature secretome which mediates thermo-pathogenicity and share a leucine rich "CL[xxxx]LHM"-motif. Considering the rapidly evolving temperature dependent-virulence and host diversity of C. lunatus, this data will be useful for designing new protection strategies.

  15. Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

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    Deng, Cecilia H; Plummer, Kim M; Jones, Darcy A B; Mesarich, Carl H; Shiller, Jason; Taranto, Adam P; Robinson, Andrew J; Kastner, Patrick; Hall, Nathan E; Templeton, Matthew D; Bowen, Joanna K

    2017-05-02

    Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host

  16. Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat

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    Yang, Fen; Jensen, Jens D.; Svensson, Birte

    2012-01-01

    Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley...... or wheat flour as the sole nutrient source to mimic the host–pathogen interaction. A gel‐based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty‐nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation...... between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase‐polymerase chain reaction in barley...

  17. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris

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    Mattanovich Diethard

    2009-06-01

    Full Text Available Abstract Background Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae. Results To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H+/glycerol transporters. Conclusion This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.

  18. Control of Secreted Protein Gene Expression and the Mammalian Secretome by the Metabolic Regulator PGC-1α.

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    Minsky, Neri; Roeder, Robert G

    2017-01-06

    Secreted proteins serve pivotal roles in the development of multicellular organisms, acting as structural matrix, extracellular enzymes, and signal molecules. However, how the secretome is regulated remains incompletely understood. Here we demonstrate, unexpectedly, that peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a critical transcriptional co-activator of metabolic gene expression, functions to down-regulate the expression of diverse genes encoding secreted molecules and extracellular matrix components to modulate the secretome. Using cell lines, primary cells, and mice, we show that both endogenous and exogenous PGC-1α down-regulate the expression of numerous genes encoding secreted molecules. Mechanistically, results obtained using mRNA stability measurements as well as intronic RNA expression analysis are consistent with a transcriptional effect of PGC-1α on the expression of genes encoding secreted proteins. Interestingly, PGC-1α requires the central heat shock response regulator heat shock factor protein 1 (HSF1) to affect some of its targets, and both factors co-reside on several target genes encoding secreted molecules in cells. Finally, using a mass spectrometric analysis of secreted proteins, we demonstrate that PGC-1α modulates the secretome of mouse embryonic fibroblasts. Our results define a link between a key pathway controlling metabolic regulation and the regulation of the mammalian secretome. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

    Directory of Open Access Journals (Sweden)

    Marlena M Wilson

    Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

  20. Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome

    DEFF Research Database (Denmark)

    Feizi, Amir; Gatto, Francesco; Uhlén, Mathias

    2017-01-01

    Protein secretory pathway in eukaryal cells is responsible for delivering functional secretory proteins. The dysfunction of this pathway causes a range of important human diseases from congenital disorders to cancer. Despite the piled-up knowledge on the molecular biology and biochemistry level...... in specific gene families of the secretory pathway. We also inspected the potential functional link between detected extreme genes and the corresponding tissues enriched secretome. As a result, the detected extreme genes showed correlation with the enrichment of the nature and number of specific post......-translational modifications in each tissue's secretome. Our findings conciliate both the housekeeping and tissue-specific nature of the protein secretory pathway, which we attribute to a fine-tuned regulation of defined gene families to support the diversity of secreted proteins and their modifications....

  1. Resolving breast cancer heterogeneity by searching reliable protein cancer biomarkers in the breast fluid secretome

    International Nuclear Information System (INIS)

    Mannello, Ferdinando; Ligi, Daniela

    2013-01-01

    One of the major goals in cancer research is to find and evaluate the early presence of biomarkers in human fluids and tissues. To resolve the complex cell heterogeneity of a tumor mass, it will be useful to characterize the intricate biomolecular composition of tumor microenvironment (the so called cancer secretome), validating secreted proteins as early biomarkers of cancer initiation and progression. This approach is not broadly applicable because of the paucity of well validated and FDA-approved biomarkers and because most of the candidate biomarkers are mainly organ-specific rather than tumor-specific. For these reasons, there is an urgent need to identify and validate a panel of biomarker combinations for early detection of human tumors. This is especially important for breast cancer, the cancer spread most worldwide among women. It is well known that patients with early diagnosed breast cancer live longer, require less extensive treatment and fare better than patients with more aggressive and/or advanced disease. In the frame of searching breast cancer biomarkers (especially using nipple aspirate fluid mirroring breast microenvironment), studies have highlighted an optimal combination of well-known biomarkers: uPA + PAI-1 + TF. When individually investigated they did not show perfect accuracy in predicting the presence of breast cancer, whereas the triple combination has been demonstrated to be highly predictive of pre-cancer and/or cancerous conditions, approaching 97-100% accuracy. Despite the heterogeneous composition of breast cancer and the difficulties to find specific breast cancer biomolecules, the noninvasive analysis of the nipple aspirate fluid secretome may significantly improve the discovery of promising biomarkers, helping also the differentiation among benign and invasive breast diseases, opening new frontiers in early oncoproteomics

  2. HCSD: the human cancer secretome database

    DEFF Research Database (Denmark)

    Feizi, Amir; Banaei-Esfahani, Amir; Nielsen, Jens

    2015-01-01

    The human cancer secretome database (HCSD) is a comprehensive database for human cancer secretome data. The cancer secretome describes proteins secreted by cancer cells and structuring information about the cancer secretome will enable further analysis of how this is related with tumor biology...... database is limiting the ability to query the increasing community knowledge. We therefore developed the Human Cancer Secretome Database (HCSD) to fulfil this gap. HCSD contains >80 000 measurements for about 7000 nonredundant human proteins collected from up to 35 high-throughput studies on 17 cancer...

  3. Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with roles in Plant Cell Wall Degradation and Virulence

    KAUST Repository

    Lakshman, Dilip; Roberts, Daniel P.; Garrett, Wesley M.; Natarajan, Savithiry S.; Darwish, Omar; Alkharouf, Nadim; Pain, Arnab; Khan, Farooq; Jambhulkar, Prashant P.; Mitra, Amitava

    2016-01-01

    Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. Here we used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions; the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups; and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

  4. Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with roles in Plant Cell Wall Degradation and Virulence

    KAUST Repository

    Lakshman, Dilip

    2016-03-28

    Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. Here we used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions; the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups; and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

  5. Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

    Science.gov (United States)

    Gómez-Mendoza, Diana Paola; Junqueira, Magno; do Vale, Luis Henrique Ferreira; Domont, Gilberto Barbosa; Ferreira Filho, Edivaldo Ximenes; Sousa, Marcelo Valle de; Ricart, Carlos André Ornelas

    2014-04-04

    The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

  6. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    Science.gov (United States)

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  7. Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Hu, Rongkuan; Huffman, Kenneth E; Chu, Michael; Zhang, Yajie; Minna, John D; Yu, Yonghao

    2016-02-05

    Lung cancer is the leading cause of cancer-related deaths for men and women in the United States, with non-small cell lung cancer (NSCLC) representing 85% of all diagnoses. Late stage detection, metastatic disease and lack of actionable biomarkers contribute to the high mortality rate. Proteins in the extracellular space are known to be critically involved in regulating every stage of the pathogenesis of lung cancer. To investigate the mechanism by which secreted proteins contribute to the pathogenesis of NSCLC, we performed quantitative secretomic analysis of two isogenic NSCLC cell lines (NCI-H1993 and NCI-H2073) and an immortalized human bronchial epithelial cell line (HBEC3-KT) as control. H1993 was derived from a chemo-naïve metastatic tumor, while H2073 was derived from the primary tumor after etoposide/cisplatin therapy. From the conditioned media of these three cell lines, we identified and quantified 2713 proteins, including a series of proteins involved in regulating inflammatory response, programmed cell death and cell motion. Gene Ontology (GO) analysis indicates that a number of proteins overexpressed in H1993 media are involved in biological processes related to cancer metastasis, including cell motion, cell-cell adhesion and cell migration. RNA interference (RNAi)-mediated knock down of a number of these proteins, including SULT2B1, CEACAM5, SPRR3, AGR2, S100P, and S100A14, leads to dramatically reduced migration of these cells. In addition, meta-analysis of survival data indicates NSCLC patients whose tumors express higher levels of several of these secreted proteins, including SULT2B1, CEACAM5, SPRR3, S100P, and S100A14, have a worse prognosis. Collectively, our results provide a potential molecular link between deregulated secretome and NSCLC cell migration/metastasis. In addition, the identification of these aberrantly secreted proteins might facilitate the development of biomarkers for early detection of this devastating disease.

  8. Lytic Polysaccharide Monooxygenases - Studies of Fungal Secretomes and Enzyme Properties

    DEFF Research Database (Denmark)

    Nekiunaite, Laura

    degradation, were also identified upstream the LPMO genes, providing evidence for a co-regulatory mechanism of LPMOs and amylolytic hydrolases. The second part of the PhD thesis is focused on understanding the binding properties of LPMOs to starch and starch mimic substrate. It was shown that LPMOs possessing...... to different substrates at the protein level. It could help to design better enzyme cocktails that increase efficiency of biomass degradation. The secretomes of A. nidulans revealed differences in growth and secretion of enzymes, depending on the type and properties of starches. A common characteristic...... conversion as they produce a wide diversity of degrading enzymes. In the first part of this PhD thesis, the secretomes of the well-known fungus Aspergillus nidulans grown on cereal and legume starches were analyzed. Secretomics is a powerful tool to unravel secretion patterns of fungi and their response...

  9. Quantitative iTRAQ secretome analysis of aspergillus niger reveals novel hydrolytic enzymes

    NARCIS (Netherlands)

    Adav, S.S.; Li, A.A.; Manavalan, A.; Punt, P.; Sze, S.K.

    2010-01-01

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was

  10. Secretome Analysis of Metarhizium anisopliae Under Submerged Conditions Using Bombyx mori Chrysalis to Induce Expression of Virulence-Related Proteins.

    Science.gov (United States)

    Rustiguel, Cynthia Barbosa; Rosa, José Cesar; Jorge, João Atílio; de Oliveira, Arthur Henrique Cavalcanti; Guimarães, Luis Henrique Souza

    2016-02-01

    The entomopathogenic fungus Metarhizium anisopliae is used to control insect pests. This species is specialized for the secretion of an enzymatic complex consisting of proteases, lipases, and chitinases related to pathogenicity and virulence. In this context, the secretomes of strains IBCB 167 and IBCB 384 of M. anisopliae var. anisopliae, grown under submerged fermentation in the presence of chrysalis as an inducer, were analyzed. Analysis of two-dimensional gels showed qualitative and quantitative differences between secreted proteins in both isolates. Around 102 protein spots were analyzed, and 76 % of the corresponding proteins identified by mass spectrometry were grouped into different classes (hydrolases, oxidases, reductases, isomerases, kinases, WSC domains, and hypothetical proteins). Thirty-three per cent of all the proteins analyzed were found to be common in both strains. Several virulence-related proteins were identified as proteases and mannosidases. Endo-N-acetyl-β-D-glucosaminidase expression was observed to be 10.14-fold higher for strain IBCB 384 than for strain IBCB 167, which may be an important contributor to the high virulence of IBCB 384 in Diatraea ssaccharalis. These results are important for elucidation of the host-pathogen relationship and the differences in virulence observed between the two strains.

  11. Secretome discovery reveals lignocellulose degradation capacity of the ectomycorrhizal fungus Paxillus involutus

    DEFF Research Database (Denmark)

    Roth, Doris; Rineau, Francois; Olsen, Peter B.

    2011-01-01

    To improve our understanding of the role ectomycorrhizal fungi play in biomass conversion, we studied the transcriptome of P. involutus grown on glass beads in extract of soil organic matter. The mycelium was used for a cDNA library screened by Transposon-Assisted Signal Trapping (TAST*) for gene...... the brown rot fungi systems. In addition, GH61 apparently acts as accessory protein both in enzymatic and in radical-based cellulolysis. * Becker et al., J. Microbial Methods, 2004, 57(1), 123-33....

  12. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  13. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    Directory of Open Access Journals (Sweden)

    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  14. Secretome Characterization and Correlation Analysis Reveal Putative Pathogenicity Mechanisms and Identify Candidate Avirulence Genes in the Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici.

    Science.gov (United States)

    Xia, Chongjing; Wang, Meinan; Cornejo, Omar E; Jiwan, Derick A; See, Deven R; Chen, Xianming

    2017-01-01

    Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most destructive diseases of wheat worldwide. Planting resistant cultivars is an effective way to control this disease, but race-specific resistance can be overcome quickly due to the rapid evolving Pst population. Studying the pathogenicity mechanisms is critical for understanding how Pst virulence changes and how to develop wheat cultivars with durable resistance to stripe rust. We re-sequenced 7 Pst isolates and included additional 7 previously sequenced isolates to represent balanced virulence/avirulence profiles for several avirulence loci in seretome analyses. We observed an uneven distribution of heterozygosity among the isolates. Secretome comparison of Pst with other rust fungi identified a large portion of species-specific secreted proteins, suggesting that they may have specific roles when interacting with the wheat host. Thirty-two effectors of Pst were identified from its secretome. We identified candidates for Avr genes corresponding to six Yr genes by correlating polymorphisms for effector genes to the virulence/avirulence profiles of the 14 Pst isolates. The putative AvYr76 was present in the avirulent isolates, but absent in the virulent isolates, suggesting that deleting the coding region of the candidate avirulence gene has produced races virulent to resistance gene Yr76 . We conclude that incorporating avirulence/virulence phenotypes into correlation analysis with variations in genomic structure and secretome, particularly presence/absence polymorphisms of effectors, is an efficient way to identify candidate Avr genes in Pst . The candidate effector genes provide a rich resource for further studies to determine the evolutionary history of Pst populations and the co-evolutionary arms race between Pst and wheat. The Avr candidates identified in this study will lead to cloning avirulence genes in Pst , which will enable us to understand molecular mechanisms

  15. Secretome of obligate intracellular Rickettsia

    Science.gov (United States)

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  16. The composition of accessory enzymes of Penicillium chrysogenum P33 revealed by secretome and synergistic effects with commercial cellulase on lignocellulose hydrolysis.

    Science.gov (United States)

    Yang, Yi; Yang, Jinshui; Liu, Jiawen; Wang, Ruonan; Liu, Liang; Wang, Fengqin; Yuan, Hongli

    2018-06-01

    Herein, we report the secretome of Penicillium chrysogenum P33 under induction of lignocellulose for the first time. A total of 356 proteins were identified, including complete cellulases and numerous hemicellulases. Supplementing a commercial cellulase with increasing dosage of P33 enzyme cocktail from 1 to 5 mg/g substrate increased the release of reducing sugars from delignified corn stover by 21.4% to 106.8%. When 50% cellulase was replaced by P33 enzyme cocktail, release of reducing sugars was 78.6% higher than with cellulase alone. Meanwhile, glucan and xylan conversion was increased by 37% and 106%, respectively. P33 enzyme cocktail also enhanced commercial cellulase hydrolysis against four different delignified lignocellulosic biomass. These findings demonstrate that mixing appropriate amount of P33 cocktail with cellulase improves polysaccharide hydrolysis, suggesting P33 enzymes have great potential for industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Mining the secretome of root-knot nematodes for cell wall modifying proteins

    NARCIS (Netherlands)

    Roze, E.H.A.

    2008-01-01

    The products of parasitism genes in nematodes must be secreted to reach their targets at the nematode-plant interface. These nematode secretory proteins are therefore recognised to play an important role in the nematode-plant interaction and as a result have been subject of intense study for years.

  18. VerSeDa: vertebrate secretome database.

    Science.gov (United States)

    Cortazar, Ana R; Oguiza, José A; Aransay, Ana M; Lavín, José L

    2017-01-01

    Based on the current tools, de novo secretome (full set of proteins secreted by an organism) prediction is a time consuming bioinformatic task that requires a multifactorial analysis in order to obtain reliable in silico predictions. Hence, to accelerate this process and offer researchers a reliable repository where secretome information can be obtained for vertebrates and model organisms, we have developed VerSeDa (Vertebrate Secretome Database). This freely available database stores information about proteins that are predicted to be secreted through the classical and non-classical mechanisms, for the wide range of vertebrate species deposited at the NCBI, UCSC and ENSEMBL sites. To our knowledge, VerSeDa is the only state-of-the-art database designed to store secretome data from multiple vertebrate genomes, thus, saving an important amount of time spent in the prediction of protein features that can be retrieved from this repository directly. VerSeDa is freely available at http://genomics.cicbiogune.es/VerSeDa/index.php. © The Author(s) 2017. Published by Oxford University Press.

  19. The Apoplastic Secretome of Trichoderma virens During Interaction With Maize Roots Shows an Inhibition of Plant Defence and Scavenging Oxidative Stress Secreted Proteins

    Directory of Open Access Journals (Sweden)

    Guillermo Nogueira-Lopez

    2018-04-01

    Full Text Available In Nature, almost every plant is colonized by fungi. Trichoderma virens is a biocontrol fungus which has the capacity to behave as an opportunistic plant endophyte. Even though many plants are colonized by this symbiont, the exact mechanisms by which Trichoderma masks its entrance into its plant host remain unknown, but likely involve the secretion of different families of proteins into the apoplast that may play crucial roles in the suppression of plant immune responses. In this study, we investigated T. virens colonization of maize roots under hydroponic conditions, evidencing inter- and intracellular colonization by the fungus and modifications in root morphology and coloration. Moreover, we show that upon host penetration, T. virens secretes into the apoplast an arsenal of proteins to facilitate inter- and intracellular colonization of maize root tissues. Using a gel-free shotgun proteomics approach, 95 and 43 secretory proteins were identified from maize and T. virens, respectively. A reduction in the maize secretome (36% was induced by T. virens, including two major groups, glycosyl hydrolases and peroxidases. Furthermore, T. virens secreted proteins were mainly involved in cell wall hydrolysis, scavenging of reactive oxygen species and secondary metabolism, as well as putative effector-like proteins. Levels of peroxidase activity were reduced in the inoculated roots, suggesting a strategy used by T. virens to manipulate host immune responses. The results provide an insight into the crosstalk in the apoplast which is essential to maintain the T. virens-plant interaction.

  20. GBM secretome induces transient transformation of human neural precursor cells.

    Science.gov (United States)

    Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

    2012-09-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

  1. Investigating Aspergillus nidulans secretome during colonisation of cork cell walls.

    Science.gov (United States)

    Martins, Isabel; Garcia, Helga; Varela, Adélia; Núñez, Oscar; Planchon, Sébastien; Galceran, Maria Teresa; Renaut, Jenny; Rebelo, Luís P N; Silva Pereira, Cristina

    2014-02-26

    Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE). Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (β-glucosidases and endo-1,5-α-l-arabinosidase). Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of β-glucosidase in cork secretome. Cork degradation by fungi remains largely overlook. Herein we aimed at understanding how A. nidulans colonise cork cell walls and how this relates to wood colonisation. To address this, the protein species consistently present in the secretome were analysed, as well as major alterations occurring in the substrate, including lignin degradation compounds being released. The obtained data demonstrate that this fungus has superficially attacked the cork cell walls apparently by

  2. The Ewing sarcoma secretome and its response to activation of Wnt/beta-catenin signaling.

    Science.gov (United States)

    Hawkins, Allegra G; Basrur, Venkatesha; da Veiga Leprevost, Felipe; Pedersen, Elisabeth; Sperring, Colin; Nesvizhskii, Alexey I; Lawlor, Elizabeth R

    2018-01-31

    Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells upregulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic

  3. Harnessing the Helminth Secretome for Therapeutic Immunomodulators

    Directory of Open Access Journals (Sweden)

    Dana Ditgen

    2014-01-01

    Full Text Available Helminths are the largest and most complex pathogens to invade and live within the human body. Since they are not able to outpace the immune system by rapid antigen variation or faster cell division or retreat into protective niches not accessible to immune effector mechanisms, their long-term survival depends on influencing and regulating the immune responses away from the mode of action most damaging to them. Immunologists have focused on the excretory and secretory products that are released by the helminths, since they can change the host environment by modulating the immune system. Here we give a brief overview of the helminth-associated immune response and the currently available helminth secretome data. We introduce some major secretome-derived immunomodulatory molecules and describe their potential mode of action. Finally, the applicability of helminth-derived therapeutic proteins in the treatment of allergic and autoimmune inflammatory disease is discussed.

  4. Comparative secretome analysis of rat stomach under different nutritional status

    Directory of Open Access Journals (Sweden)

    Lucia L. Senin

    2015-06-01

    Full Text Available The fact that gastric surgery is at the moment the most effective treatment to fight against obesity highlights the relevance of gastric derived proteins as potential targets to treat this pathology. Taking advantage of a previously established gastric explant model for endocrine studies, the proteomic analysis of gastric secretome was performed. To validate this gastric explant system for proteomic analysis, the identification of ghrelin, a classical gastric derived peptide, was performed by MS. In addition, the differential analysis of gastric secretomes under differential nutritional status (control feeding vs fasting vs re-feeding was performed. The MS identified proteins are showed in the present manuscript. The data supplied in this article is related to the research article entitled “Comparative secretome analysis of rat stomach under different nutritional status” [1].

  5. Secretome of Aspergillus oryzae in Shaoxing rice wine koji.

    Science.gov (United States)

    Zhang, Bo; Guan, Zheng-Bing; Cao, Yu; Xie, Guang-Fa; Lu, Jian

    2012-04-16

    Shaoxing rice wine is the most famous and representative Chinese rice wine. Aspergillus oryzae SU16 is used in the manufacture of koji, the Shaoxing rice wine starter culture. In the current study, a comprehensive analysis of the secretome profile of A. oryzae SU16 in Shaoxing rice wine koji was performed for the first time. The proteomic analysis for the identification of the secretory proteins was done using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry based on the annotated A. oryzae genome sequence. A total of 41 unique proteins were identified from the secretome. These proteins included 17 extracellular proteins following the classical secretory pathway, and 10 extracellular proteins putatively secreted by the non-classical secretory pathway. The present secretome profile greatly differed from previous reports on A. oryzae growing in other solid-state nutrient sources. Several new secretory or putative secretory proteins were also found. These proteomic data will significantly aid the advancement of research on the secretome of A. oryzae, especially in solid-state cultures, and in elucidating the production process mechanism of Shaoxing rice wine koji. The findings may promote the technological development and innovation of the Shaoxing rice wine industry. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Cigarette smoke alters the secretome of lung epithelial cells.

    Science.gov (United States)

    Mossina, Alessandra; Lukas, Christina; Merl-Pham, Juliane; Uhl, Franziska E; Mutze, Kathrin; Schamberger, Andrea; Staab-Weijnitz, Claudia; Jia, Jie; Yildirim, Ali Ö; Königshoff, Melanie; Hauck, Stefanie M; Eickelberg, Oliver; Meiners, Silke

    2017-01-01

    Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Characterization of Regenerative Phenotype of Unrestricted Somatic Stem Cells (USSC) from Human Umbilical Cord Blood (hUCB) by Functional Secretome Analysis*

    Science.gov (United States)

    Schira, Jessica; Falkenberg, Heiner; Hendricks, Marion; Waldera-Lupa, Daniel M.; Kögler, Gesine; Meyer, Helmut E.; Müller, Hans Werner; Stühler, Kai

    2015-01-01

    Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes. PMID:26183719

  8. Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

    Czech Academy of Sciences Publication Activity Database

    Hafidh, Said; Potěšil, D.; Fíla, Jan; Čapková, Věra; Zdráhal, Z.; Honys, David

    2016-01-01

    Roč. 17, MAY 3 (2016), č. článku 81. ISSN 1465-6906 R&D Projects: GA ČR GA15-22720S; GA ČR(CZ) GA14-32292S; GA ČR(CZ) GA15-16050S; GA MŠk(CZ) LD14109; GA MŠk(CZ) LM2015043; GA MŠk(CZ) ED1.1.00/02.0068; GA MŠk(CZ) LQ1601 Institutional support: RVO:61389030 Keywords : Protein secretion * Pollen tube guidance * Cell-cell signaling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 11.313, year: 2015

  9. Plasmodium falciparum secretome in erythrocyte and beyond

    Directory of Open Access Journals (Sweden)

    Rani eSoni

    2016-02-01

    Full Text Available Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for development of novel anti-malarial therapies.

  10. Comparative and bioinformatics analyses of pathogenic bacterial secretomes identified by mass spectrometry in Burkholderia species.

    Science.gov (United States)

    Nguyen, Thao Thi; Chon, Tae-Soo; Kim, Jaehan; Seo, Young-Su; Heo, Muyoung

    2017-07-01

    Secreted proteins (secretomes) play crucial roles during bacterial pathogenesis in both plant and human hosts. The identification and characterization of secretomes in the two plant pathogens Burkholderia glumae BGR1 and B. gladioli BSR3, which cause diseases in rice such as seedling blight, panicle blight, and grain rot, are important steps to not only understand the disease-causing mechanisms but also find remedies for the diseases. Here, we identified two datasets of secretomes in B. glumae BGR1 and B. gladioli BSR3, which consist of 118 and 111 proteins, respectively, using mass spectrometry approach and literature curation. Next, we characterized the functional properties, potential secretion pathways and sequence information properties of secretomes of two plant pathogens in a comparative analysis by various computational approaches. The ratio of potential non-classically secreted proteins (NCSPs) to classically secreted proteins (CSPs) in B. glumae BGR1 was greater than that in B. gladioli BSR3. For CSPs, the putative hydrophobic regions (PHRs) which are essential for secretion process of CSPs were screened in detail at their N-terminal sequences using hidden Markov model (HMM)-based method. Total 31 pairs of homologous proteins in two bacterial secretomes were indicated based on the global alignment (identity ≥ 70%). Our results may facilitate the understanding of the species-specific features of secretomes in two plant pathogenic Burkholderia species.

  11. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

    DEFF Research Database (Denmark)

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affe...

  12. Secretome analysis of the fungus Trichoderma harzianum grown on cellulose.

    Science.gov (United States)

    Do Vale, Luis H F; Gómez-Mendoza, Diana P; Kim, Min-Sik; Pandey, Akhilesh; Ricart, Carlos A O; Ximenes F Filho, Edivaldo; Sousa, Marcelo V

    2012-08-01

    Trichoderma harzianum is a mycoparasitic filamentous fungus that produces and secretes a wide range of extracellular hydrolytic enzymes used in cell wall degradation. Due to its potential in biomass conversion, T. harzianum draws great attention from biofuel and biocontrol industries and research. Here, we report an extensive secretome analysis of T. harzianum. The fungus was grown on cellulose medium, and its secretome was analyzed by a combination of enzymology, 2DE, MALDI-MS and -MS/MS (Autoflex II), and LC-MS/MS (LTQ-Orbitrap XL). A total of 56 proteins were identified using high-resolution MS. Interestingly, although cellulases were found, the major hydrolytic enzymes secreted in the cellulose medium were chitinases and endochitinases, which may reflect the biocontrol feature of T. harzianum. The glycoside hydrolase family, including chitinases (EC 3.2.1.14), endo-N-acetylglucosaminidases (EC 3.2.1.96), hexosaminidases (EC 3.2.1.52), galactosidases (EC 3.2.1.23), xylanases (EC 3.2.1.8), exo-1,3-glucanases (EC 3.2.1.58), endoglucanases (EC 3.2.1.4), xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), N-acetylhexosaminidases (EC 3.2.1.52), and other enzymes represented 51.36% of the total secretome. Few representatives were classified in the protease family (8.90%). Others (17.60%) are mostly intracellular proteins. A considerable part of the secretome was composed of hypothetical proteins (22.14%), probably because of the absence of an annotated T. harzianum genome. The T. harzianum secretome composition highlights the importance of this fungus as a rich source of hydrolytic enzymes for bioconversion and biocontrol applications. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Methodologies and perspectives of proteomics applied to filamentous fungi: from sample preparation to secretome analysis.

    Science.gov (United States)

    Bianco, Linda; Perrotta, Gaetano

    2015-03-12

    Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.

  14. Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

    Science.gov (United States)

    Bianco, Linda; Perrotta, Gaetano

    2015-01-01

    Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation. PMID:25775160

  15. Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources

    OpenAIRE

    Sunil S. Adav; Anita Ravindran; Siu Kwan Sze

    2015-01-01

    Here, we provide data related to the research article entitled ?Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes? by Adav et al. (J. Proteomics (2015) [1]). Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using i...

  16. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

    Directory of Open Access Journals (Sweden)

    Thomas R. Cox

    2015-12-01

    Full Text Available The cancer secretome includes all of the macromolecules secreted by cells into their microenvironment. Cancer cell secretomes are significantly different to that of normal cells reflecting the changes that normal cells have undergone during their transition to malignancy. More importantly, cancer secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397 and the complete proteomic, bioinformatic and biological analyses are reported in Cox et al. (2015 [1].

  17. Characterization of the secretomes of two vibrios pathogenic to mollusks.

    Directory of Open Access Journals (Sweden)

    Stéphanie Madec

    Full Text Available Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion. Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively. Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease. For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.

  18. Angiogenin induces modifications in the astrocyte secretome: relevance to amyotrophic lateral sclerosis.

    Science.gov (United States)

    Skorupa, Alexandra; Urbach, Serge; Vigy, Oana; King, Matthew A; Chaumont-Dubel, Séverine; Prehn, Jochen H M; Marin, Philippe

    2013-10-08

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting lower and upper motoneurons. Recent studies have shown that both motor neurons and non-neuronal neighbouring cells such as astrocytes and microglia contribute to disease pathology. Loss-of-function mutations in the angiogenin (ANG) gene have been identified in ALS patients. Angiogenin is enriched in motor neurons and exerts neuroprotective effects in vitro and in vivo. We have recently shown that motoneurons secrete angiogenin, and that secreted angiogenin is exclusively taken up by astrocytes, suggesting a paracrine mechanism of neuroprotection. To gain insights into astrocyte effectors of angiogenin-induced neuroprotection, we examined alterations in the astrocyte secretome induced by angiogenin treatment using quantitative proteomics based on Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC). We identified 2128 proteins in conditioned media from primary cultured mouse astrocytes, including 1247 putative secreted proteins. Of these, 60 proteins showed significant regulation of secretion in response to angiogenin stimulation. Regulated proteins include chemokines and cytokines, proteases and protease inhibitors as well as proteins involved in reorganising the extracellular matrix. In conclusion, this proteomic analysis increases our knowledge of the astrocyte secretome and reveals potential molecular substrates underlying the paracrine, neuroprotective effects of angiogenin. This study provides the most extensive list of astrocyte-secreted proteins available and reveals novel potential molecular substrates of astrocyte-neuron communication. It also identifies a set of astrocyte-derived proteins that might slow down ALS disease progression. It should be relevant to a large readership of neuroscientists and clinicians, in particular those with an interest in the physiological and pathological roles of astrocytes and in the molecular and cellular mechanisms underlying

  19. The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves.

    Science.gov (United States)

    Chuc-Uc, José; Brito-Argáez, Ligia; Canto-Canché, Blondy; Tzec-Simá, Miguel; Rodríguez-García, Cecilia; Peraza-Echeverría, Leticia; Peraza-Echeverría, Santy; James-Kay, Andrew; Cruz-Cruz, Carlos Alberto; Peña-Rodríguez, Luis Manuel; Islas-Flores, Ignacio

    2011-06-01

    The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  20. Analysis of secretome of breast cancer cell line with an optimized semi-shotgun method

    International Nuclear Information System (INIS)

    Tang Xiaorong; Yao Ling; Chen Keying; Hu Xiaofang; Xu Lisa; Fan Chunhai

    2009-01-01

    Secretome, the totality of secreted proteins, is viewed as a promising pool of candidate cancer biomarkers. Simple and reliable methods for identifying secreted proteins are highly desired. We used an optimized semi-shotgun liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) method to analyze the secretome of breast cancer cell line MDA-MB-231. A total of 464 proteins were identified. About 63% of the proteins were classified as secreted proteins, including many promising breast cancer biomarkers, which were thought to be correlated with tumorigenesis, tumor development and metastasis. These results suggest that the optimized method may be a powerful strategy for cell line secretome profiling, and can be used to find potential cancer biomarkers with great clinical significance. (authors)

  1. Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome.

    Science.gov (United States)

    Aragonès, Gemma; Auguet, Teresa; Guiu-Jurado, Esther; Berlanga, Alba; Curriu, Marta; Martinez, Salomé; Alibalic, Ajla; Aguilar, Carmen; Hernández, Esteban; Camara, María-Luisa; Canela, Núria; Herrero, Pol; Ruyra, Xavier; Martín-Paredero, Vicente; Richart, Cristóbal

    2016-03-04

    Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

  2. Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

    Science.gov (United States)

    Witzke, Kathrin E; Rosowski, Kristin; Müller, Christian; Ahrens, Maike; Eisenacher, Martin; Megger, Dominik A; Knobloch, Jürgen; Koch, Andrea; Bracht, Thilo; Sitek, Barbara

    2017-01-06

    Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

  3. Comparative secretome analysis suggests low plant cell wall degrading capacity in Frankia symbionts

    Directory of Open Access Journals (Sweden)

    Normand Philippe

    2008-01-01

    Full Text Available Abstract Background Frankia sp. strains, the nitrogen-fixing facultative endosymbionts of actinorhizal plants, have long been proposed to secrete hydrolytic enzymes such as cellulases, pectinases, and proteases that may contribute to plant root penetration and formation of symbiotic root nodules. These or other secreted proteins might logically be involved in the as yet unknown molecular interactions between Frankia and their host plants. We compared the genome-based secretomes of three Frankia strains representing diverse host specificities. Signal peptide detection algorithms were used to predict the individual secretomes of each strain, and the set of secreted proteins shared among the strains, termed the core Frankia secretome. Proteins in the core secretome may be involved in the actinorhizal symbiosis. Results The Frankia genomes have conserved Sec (general secretory and Tat (twin arginine translocase secretion systems. The potential secretome of each Frankia strain comprised 4–5% of the total proteome, a lower percentage than that found in the genomes of other actinobacteria, legume endosymbionts, and plant pathogens. Hydrolytic enzymes made up only a small fraction of the total number of predicted secreted proteins in each strain. Surprisingly, polysaccharide-degrading enzymes were few in number, especially in strain CcI3, with more esterolytic, lipolytic and proteolytic enzymes having signal peptides. A total of 161 orthologous proteins belong to the core Frankia secretome. Of these, 52 also lack homologs in closely related actinobacteria, and are termed "Frankia-specific." The genes encoding these conserved secreted proteins are often clustered near secretion machinery genes. Conclusion The predicted secretomes of Frankia sp. are relatively small and include few hydrolases, which could reflect adaptation to a symbiotic lifestyle. There are no well-conserved secreted polysaccharide-degrading enzymes present in all three Frankia

  4. Secretome-based Manganese(II) Oxidation by Filamentous Ascomycete Fungi

    Science.gov (United States)

    Zeiner, C. A.; Purvine, S.; Zink, E.; Paša-Tolić, L.; Chaput, D.; Wu, S.; Santelli, C. M.; Hansel, C. M.

    2017-12-01

    Manganese (Mn) oxides are among the strongest oxidants in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and Mn peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Through a combination of chemical and in-gel assays, bulk mass spectrometry, and iTRAQ proteomics, we demonstrate enzymatic Mn(II) oxidation in the secretomes of three phylogenetically diverse Ascomycetes that were isolated from Mn-laden sediments. Candidate Mn(II)-oxidizing enzymes were species-specific and included bilirubin oxidase and tyrosinase in Stagonospora sp. SRC1lsM3a, GMC oxidoreductase in Paraconiothyrium sporulosum AP3s5-JAC2a, and FAD-binding oxidoreductases in Pyrenochaeta sp. DS3sAY3a. These findings were supported by full proteomic characterization of the secretomes, which revealed a lack of Mn, lignin, and versatile peroxidases in these Ascomycetes but a substantially higher proportion of LMCOs and GMC oxidoreductases compared to wood-rot Basidiomycetes. We also identified the potential for indirect enzymatic Mn(II) oxidation by hydroxyl radical, as the secretomes were rich in diverse lignocellulose-degrading enzymes that could participate in Fenton chemistry. A link between Mn(II) oxidation and carbon oxidation analogous to white-rot Basidiomycetes remains unknown in these Ascomycetes. Interestingly, growth rates on rich medium were unaffected by the presence of Mn(II), and the production of Mn(II)-oxidizing proteins in the secretome was constitutive and not inducible by Mn(II). Thus, no physiological benefit of Mn(II) oxidation in these Ascomycetes has yet been identified, and Mn(II) oxidation appears to be a side reaction. Future work will explore the lignin-degrading capacity of

  5. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

    DEFF Research Database (Denmark)

    Cox, T.R.; Schoof, Erwin; Gartland, A.

    2015-01-01

    secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic...... and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397...

  6. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

    Science.gov (United States)

    Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

    2012-01-10

    The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  7. Secretome of fungus-infected aphids documents high pathogen activity and weak host response

    DEFF Research Database (Denmark)

    Grell, Morten Nedergaard; Jensen, Annette Bruun; Olsen, Peter B.

    2011-01-01

    Discovery of novel secretome proteins contributes to the understanding of host-pathogen interactions. Here we report a rich diversity of secreted proteins from the interaction between grain aphids (host, insect order Hemiptera) and fungi of the order Entomophthorales (insect pathogens), made...

  8. Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

    Science.gov (United States)

    Staats, Charley Christian; Junges, Angela; Guedes, Rafael Lucas Muniz; Thompson, Claudia Elizabeth; de Morais, Guilherme Loss; Boldo, Juliano Tomazzoni; de Almeida, Luiz Gonzaga Paula; Andreis, Fábio Carrer; Gerber, Alexandra Lehmkuhl; Sbaraini, Nicolau; da Paixão, Rana Louise de Andrade; Broetto, Leonardo; Landell, Melissa; Santi, Lucélia; Beys-da-Silva, Walter Orlando; Silveira, Carolina Pereira; Serrano, Thaiane Rispoli; de Oliveira, Eder Silva; Kmetzsch, Lívia; Vainstein, Marilene Henning; de Vasconcelos, Ana Tereza Ribeiro; Schrank, Augusto

    2014-09-29

    Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

  9. Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest.

    Science.gov (United States)

    Cologna, Nicholas de Mojana di; Gómez-Mendoza, Diana Paola; Zanoelo, Fabiana Fonseca; Giannesi, Giovana Cristina; Guimarães, Nelciele Cavalieri de Alencar; Moreira, Leonora Rios de Souza; Filho, Edivaldo Ximenes Ferreira; Ricart, Carlos André Ornelas

    2018-02-01

    Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Analytical and computational approaches to define the Aspergillus niger secretome

    Energy Technology Data Exchange (ETDEWEB)

    Tsang, Adrian; Butler, Gregory D.; Powlowski, Justin; Panisko, Ellen A.; Baker, Scott E.

    2009-03-01

    We used computational and mass spectrometric approaches to characterize the Aspergillus niger secretome. The 11,200 gene models predicted in the genome of A. niger strain ATCC 1015 were the data source for the analysis. Depending on the computational methods used, 691 to 881 proteins were predicted to be secreted proteins. We cultured A. niger in six different media and analyzed the extracellular proteins produced using mass spectrometry. A total of 222 proteins were identified, with 39 proteins expressed under all six conditions and 74 proteins expressed under only one condition. The secreted proteins identified by mass spectrometry were used to guide the correction of about 20 gene models. Additional analysis focused on extracellular enzymes of interest for biomass processing. Of the 63 glycoside hydrolases predicted to be capable of hydrolyzing cellulose, hemicellulose or pectin, 94% of the exo-acting enzymes and only 18% of the endo-acting enzymes were experimentally detected.

  11. Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

    Science.gov (United States)

    Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2016-01-01

    Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

  12. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

    NARCIS (Netherlands)

    Braaksma, M.; Martens-Uzunova, E.S.; Punt, P.J.; Schaap, P.J.

    2010-01-01

    Background: The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using

  13. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

    NARCIS (Netherlands)

    Braaksma, M.; Martens-Uzunova, E.S.; Punt, P.J.; Schaap, P.J.

    2010-01-01

    BACKGROUND: The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using

  14. Comparative secretome analysis of rat stomach under different nutritional status.

    Science.gov (United States)

    Senin, Lucia L; Roca-Rivada, Arturo; Castelao, Cecilia; Alonso, Jana; Folgueira, Cintia; Casanueva, Felipe F; Pardo, Maria; Seoane, Luisa M

    2015-02-26

    Obesity is a major public health threat for many industrialised countries. Bariatric surgery is the most effective treatment against obesity, suggesting that gut derived signals are crucial for energy balance regulation. Several descriptive studies have proven the presence of gastric endogenous systems that modulate energy homeostasis; however, these systems and the interactions between them are still not well known. In the present study, we show for the first time the comparative 2-DE gastric secretome analysis under different nutritional status. We have identified 38 differently secreted proteins by comparing stomach secretomes from tissue explant cultures of rats under feeding, fasting and re-feeding conditions. Among the proteins identified, glyceraldehyde-3-phosphate dehydrogenase was found to be more abundant in gastric secretome and plasma after re-feeding, and downregulated in obesity. Additionally, two calponin-1 species were decreased in feeding state, and other were modulated by nutritional and metabolic conditions. These and other secreted proteins identified in this work may be considered as potential gastrokines implicated in food intake regulation. The present work has an important impact in the field of obesity, especially in the regulation of body weight maintenance by the stomach. Nowadays, the most effective treatment in the fight against obesity is bariatric surgery, which suggests that stomach derived signals might be crucial for the regulation of the energy homeostasis. However, until now, the knowledge about the gastrokines and its mechanism of action has been poorly elucidated. In the present work, we had updated a previously validated explant secretion model for proteomic studies; this analysis allowed us, for the first time, to study the gastric secretome without interferences from other organs. We had identified 38 differently secreted proteins comparing ex vivo cultured stomachs from rats under feeding, fasting and re-feeding regimes

  15. A rapidly evolving secretome builds and patterns a sea shell

    Directory of Open Access Journals (Sweden)

    Green Kathryn

    2006-11-01

    Full Text Available Abstract Background Instructions to fabricate mineralized structures with distinct nanoscale architectures, such as seashells and coral and vertebrate skeletons, are encoded in the genomes of a wide variety of animals. In mollusks, the mantle is responsible for the extracellular production of the shell, directing the ordered biomineralization of CaCO3 and the deposition of architectural and color patterns. The evolutionary origins of the ability to synthesize calcified structures across various metazoan taxa remain obscure, with only a small number of protein families identified from molluskan shells. The recent sequencing of a wide range of metazoan genomes coupled with the analysis of gene expression in non-model animals has allowed us to investigate the evolution and process of biomineralization in gastropod mollusks. Results Here we show that over 25% of the genes expressed in the mantle of the vetigastropod Haliotis asinina encode secreted proteins, indicating that hundreds of proteins are likely to be contributing to shell fabrication and patterning. Almost 85% of the secretome encodes novel proteins; remarkably, only 19% of these have identifiable homologues in the full genome of the patellogastropod Lottia scutum. The spatial expression profiles of mantle genes that belong to the secretome is restricted to discrete mantle zones, with each zone responsible for the fabrication of one of the structural layers of the shell. Patterned expression of a subset of genes along the length of the mantle is indicative of roles in shell ornamentation. For example, Has-sometsuke maps precisely to pigmentation patterns in the shell, providing the first case of a gene product to be involved in molluskan shell pigmentation. We also describe the expression of two novel genes involved in nacre (mother of pearl deposition. Conclusion The unexpected complexity and evolvability of this secretome and the modular design of the molluskan mantle enables

  16. Proteomic Studies of Cholangiocarcinoma and Hepatocellular Carcinoma Cell Secretomes

    Directory of Open Access Journals (Sweden)

    Chantragan Srisomsap

    2010-01-01

    Full Text Available Cholangiocarcinoma (CCA and hepatocellular carcinoma (HCC occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1 and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2, laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

  17. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

    OpenAIRE

    Braaksma, Machtelt; Martens-Uzunova, Elena S; Punt, Peter J; Schaap, Peter J

    2010-01-01

    Abstract Background The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actuall...

  18. Bone Marrow Stromal Antigen 2 Is a Novel Plasma Biomarker and Prognosticator for Colorectal Carcinoma: A Secretome-Based Verification Study

    Directory of Open Access Journals (Sweden)

    Sum-Fu Chiang

    2015-01-01

    Full Text Available Background. The cancer cell secretome has been recognized as a valuable reservoir for identifying novel serum/plasma biomarkers for different cancers, including colorectal cancer (CRC. This study aimed to verify four CRC cell-secreted proteins (tumor-associated calcium signal transducer 2/trophoblast cell surface antigen 2 (TACSTD2/TROP2, tetraspanin-6 (TSPAN6, bone marrow stromal antigen 2 (BST2, and tumor necrosis factor receptor superfamily member 16 (NGFR as potential plasma CRC biomarkers. Methods. The study population comprises 152 CRC patients and 152 controls. Target protein levels in plasma and tissue samples were assessed by ELISA and immunohistochemistry, respectively. Results. Among the four candidate proteins examined by ELISA in a small sample set, only BST2 showed significantly elevated plasma levels in CRC patients versus controls. Immunohistochemical analysis revealed the overexpression of BST2 in CRC tissues, and higher BST2 expression levels correlated with poorer 5-year survival (46.47% versus 65.57%; p=0.044. Further verification confirmed the elevated plasma BST2 levels in CRC patients (2.35 ± 0.13 ng/mL versus controls (1.04 ± 0.03 ng/mL (p<0.01, with an area under the ROC curve (AUC being 0.858 comparable to that of CEA (0.867. Conclusion. BST2, a membrane protein selectively detected in CRC cell secretome, may be a novel plasma biomarker and prognosticator for CRC.

  19. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Directory of Open Access Journals (Sweden)

    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  20. Secretome data from Trichoderma reesei and Aspergillus niger cultivated in submerged and sequential fermentation methods

    Directory of Open Access Journals (Sweden)

    Camila Florencio

    2016-09-01

    Full Text Available The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc., glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in “Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis” C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016 [1]. Keywords: Tricoderma reesei, Aspergillus Niger, Enzyme Production, Secretome

  1. Optical tweezers reveal how proteins alter replication

    Science.gov (United States)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  2. Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing

    Energy Technology Data Exchange (ETDEWEB)

    Dumortier, Jerome; Streblow, Daniel N.; Moses, Ashlee V.; Jacobs, Jon M.; Kreklywich, Craig N.; Camp, David G.; Smith, Richard D.; Orloff, Susan L.; Nelson, Jay

    2008-07-01

    Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.

  3. Comparative Analysis of Secretome Profiles of Manganese(II-Oxidizing Ascomycete Fungi.

    Directory of Open Access Journals (Sweden)

    Carolyn A Zeiner

    Full Text Available Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a. We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

  4. Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi

    Energy Technology Data Exchange (ETDEWEB)

    Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.; Paša-Tolić, Ljiljana; Chaput, Dominique L.; Haridas, Sajeet; Wu, Si; LaButti, Kurt; Grigoriev, Igor V.; Henrissat, Bernard; Santelli, Cara M.; Hansel, Colleen M.; Pöggeler, Stefanie

    2016-07-19

    Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

  5. Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata

    Directory of Open Access Journals (Sweden)

    Shiels Brian R

    2010-01-01

    Full Text Available Abstract Background Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. Results Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. Conclusions

  6. Comparative analysis of the in vitro and in planta secretomes from Mycosphaerella fijiensis isolates.

    Science.gov (United States)

    Escobar-Tovar, Lina; Guzmán-Quesada, Mauricio; Sandoval-Fernández, Jorge A; Gómez-Lim, Miguel A

    2015-06-01

    Black Sigatoka, a devastating disease of bananas and plantains worldwide, is caused by the fungus Mycosphaerella fijiensis. Several banana cultivars such as 'Yangambi Km 5' and Calcutta IV, have been known to be resistant to the fungus, but the resistance has been broken in 'Yangambi Km 5' in Costa Rica. Since the resistance of this variety still persists in Mexico, the aim of this study was to compare the in vitro and in planta secretomes from two avirulent and virulent M. fijiensis isolates using proteomics and bioinformatics approaches. We aimed to identify differentially expressed proteins in fungal isolates that differ in pathogenicity and that might be responsible for breaking the resistance in 'Yangambi Km 5'. We were able to identify 90 protein spots in the secretomes of fungal isolates encoding 42 unique proteins and 35 differential spots between them. Proteins involved in carbohydrate transport and metabolism were more prevalent. Several proteases, pathogenicity-related, ROS detoxification and unknown proteins were also highly or specifically expressed by the virulent isolate in vitro or during in planta infection. An unknown protein representing a virulence factor candidate was also identified. These results demonstrated that the secretome reflects major differences between both M. fijiensis isolates. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  7. Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

    Directory of Open Access Journals (Sweden)

    Jackson Champer

    2016-01-01

    Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

  8. Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential.

    Science.gov (United States)

    Sultan, Abida; Frisvad, Jens C; Andersen, Birgit; Svensson, Birte; Finnie, Christine

    2017-10-03

    The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

    Science.gov (United States)

    Tsumagari, Kazuya; Shirakabe, Kyoko; Ogura, Mayu; Sato, Fuminori; Ishihama, Yasushi; Sehara-Fujisawa, Atsuko

    2017-02-01

    Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  10. Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482.

    Directory of Open Access Journals (Sweden)

    Warren W Wakarchuk

    Full Text Available The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes. Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC, as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

  11. Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs.

    Science.gov (United States)

    Winger, A M; Heazlewood, J L; Chan, L J G; Petzold, C J; Permaul, K; Singh, S

    2014-11-01

    Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.

  12. Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2010-01-01

    During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative...... proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics...... of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188...

  13. Molecular Therapy for Degenerative Disc Disease: Clues from Secretome Analysis of the Notochordal Cell-Rich Nucleus Pulposus

    Science.gov (United States)

    Matta, Ajay; Karim, M. Zia; Isenman, David E.; Erwin, W. Mark

    2017-01-01

    Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFβ- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFβ1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFβ1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc. PMID:28358123

  14. Acidithiobacillus thiooxidans secretome containing a newly described lipoprotein Licanantase enhances chalcopyrite bioleaching rate

    Science.gov (United States)

    Bobadilla Fazzini, Roberto A.; Levican, Gloria

    2010-01-01

    The nature of the mineral–bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743–2747, 1998). On this respect, despite Acidithiobacilli—a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)—has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788–789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492–493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-3063-8) contains supplementary material, which is available to authorized users. PMID:21191788

  15. Type conversion of secretomes in a 3D TAM2 and HCC cell co-culture system and functional importance of CXCL2 in HCC.

    Science.gov (United States)

    Lu, Yu; Li, Shan; Ma, Liping; Li, Yan; Zhang, Xiaolian; Peng, Qiliu; Mo, Cuiju; Huang, Li; Qin, Xue; Liu, Yinkun

    2016-04-27

    Macrophages play important roles in the tumor microenvironment, driving cancer progression and metastasis, particularly in hepatocellular carcinoma (HCC). However, few studies have assessed the exact secretome composition in HCC. In the present study, the impact of different phenotype of macrophages on HCC cells was investigated. Alternatively activated macrophages (M2) were found to significantly increase the proliferation, migration, and invasion abilities of SMMC7721 cells (all P cultured with SMMC7721 cells to reconstruct the tumor microenvironment. Conditioned medium from 3D single cultures of M2, SMMC7721 cells, and their co-culture system were analyzed using quantitative proteomics via iTRAQ labeling combined with mass spectrometric analysis. Secretome analysis revealed a total of 159 differential secreted proteins in the co-culture system compared to the single culture systems, with 63 being up-regulated (>1.3-fold) and 96 down-regulated (culture system and HCC tissues, and was selected for further investigation. Functional effects data suggested that recombinant human CXCL2 significantly enhanced the migration, invasion ability of SMMC7721 cells, and weakened adhesion ability. While CXCL2 neutralization and CXCR2 blockage significantly inhibited the effects of CXCL2 on SMMC7721 cells, indicating that CXCL2 may play pivotal role in HCC metastasis.

  16. Construction and Screening of a Lentiviral Secretome Library.

    Science.gov (United States)

    Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

    2017-06-22

    Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Secretome Analysis of Lipid-Induced Insulin Resistance in Skeletal Muscle Cells by a Combined Experimental and Bioinformatics Workflow

    DEFF Research Database (Denmark)

    Deshmukh, Atul S; Cox, Juergen; Jensen, Lars Juhl

    2015-01-01

    , in principle, allows an unbiased and comprehensive analysis of cellular secretomes; however, the distinction of bona fide secreted proteins from proteins released upon lysis of a small fraction of dying cells remains challenging. Here we applied highly sensitive MS and streamlined bioinformatics to analyze......-resistant conditions. Our study demonstrates an efficient combined experimental and bioinformatics workflow to identify putative secreted proteins from insulin-resistant skeletal muscle cells, which could easily be adapted to other cellular models....

  18. Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources

    Directory of Open Access Journals (Sweden)

    Sunil S. Adav

    2015-06-01

    Full Text Available Here, we provide data related to the research article entitled “Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes” by Adav et al. (J. Proteomics (2015 [1]. Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ. The data presented here represents the detailed comparative abundances of diverse groups of biomass hydrolyzing enzymes including cellulases, hemicellulases, lignin degrading enzymes, and peptidases and proteases; and their post translational modification like deamidation.

  19. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data

    Directory of Open Access Journals (Sweden)

    Martens-Uzunova Elena S

    2010-10-01

    Full Text Available Abstract Background The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. Results A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. Conclusions We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

  20. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data.

    Science.gov (United States)

    Braaksma, Machtelt; Martens-Uzunova, Elena S; Punt, Peter J; Schaap, Peter J

    2010-10-19

    The ecological niche occupied by a fungal species, its pathogenicity and its usefulness as a microbial cell factory to a large degree depends on its secretome. Protein secretion usually requires the presence of a N-terminal signal peptide (SP) and by scanning for this feature using available highly accurate SP-prediction tools, the fraction of potentially secreted proteins can be directly predicted. However, prediction of a SP does not guarantee that the protein is actually secreted and current in silico prediction methods suffer from gene-model errors introduced during genome annotation. A majority rule based classifier that also evaluates signal peptide predictions from the best homologs of three neighbouring Aspergillus species was developed to create an improved list of potential signal peptide containing proteins encoded by the Aspergillus niger genome. As a complement to these in silico predictions, the secretome associated with growth and upon carbon source depletion was determined using a shotgun proteomics approach. Overall, some 200 proteins with a predicted signal peptide were identified to be secreted proteins. Concordant changes in the secretome state were observed as a response to changes in growth/culture conditions. Additionally, two proteins secreted via a non-classical route operating in A. niger were identified. We were able to improve the in silico inventory of A. niger secretory proteins by combining different gene-model predictions from neighbouring Aspergilli and thereby avoiding prediction conflicts associated with inaccurate gene-models. The expected accuracy of signal peptide prediction for proteins that lack homologous sequences in the proteomes of related species is 85%. An experimental validation of the predicted proteome confirmed in silico predictions.

  1. Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.

    Science.gov (United States)

    Meinhardt, Lyndel W; Costa, Gustavo Gilson Lacerda; Thomazella, Daniela P T; Teixeira, Paulo José P L; Carazzolle, Marcelo Falsarella; Schuster, Stephan C; Carlson, John E; Guiltinan, Mark J; Mieczkowski, Piotr; Farmer, Andrew; Ramaraj, Thiruvarangan; Crozier, Jayne; Davis, Robert E; Shao, Jonathan; Melnick, Rachel L; Pereira, Gonçalo A G; Bailey, Bryan A

    2014-02-27

    The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase. Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the

  2. Mononuclear cell secretome protects from experimental autoimmune myocarditis.

    Science.gov (United States)

    Hoetzenecker, Konrad; Zimmermann, Matthias; Hoetzenecker, Wolfram; Schweiger, Thomas; Kollmann, Dagmar; Mildner, Michael; Hegedus, Balazs; Mitterbauer, Andreas; Hacker, Stefan; Birner, Peter; Gabriel, Christian; Gyöngyösi, Mariann; Blyszczuk, Przemyslaw; Eriksson, Urs; Ankersmit, Hendrik Jan

    2015-03-14

    Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Cardiology.

  3. Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger.

    Science.gov (United States)

    Krijgsheld, Pauline; Altelaar, A F Maarten; Post, Harm; Ringrose, Jeffrey H; Müller, Wally H; Heck, Albert J R; Wösten, Han A B

    2012-05-04

    Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

  4. High and Low Doses of Ionizing Radiation Induce Different Secretome Profiles in a Human Skin Model

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Matzke, Melissa M.; Schepmoes, Athena A.; Moore, Ronald J.; Webb-Robertson, Bobbie-Jo M.; Hu, Zeping; Monroe, Matthew E.; Qian, Weijun; Smith, Richard D.; Morgan, William F.

    2014-03-18

    It is postulated that secreted soluble factors are important contributors of bystander effect and adaptive responses observed in low dose ionizing radiation. Using multidimensional liquid chromatography-mass spectrometry based proteomics, we quantified the changes of skin tissue secretome – the proteins secreted from a full thickness, reconstituted 3-dimensional skin tissue model 48 hr after exposure to 3, 10 and 200 cGy of X-rays. Overall, 135 proteins showed statistical significant difference between the sham (0 cGy) and any of the irradiated groups (3, 10 or 200 cGy) on the basis of Dunnett adjusted t-test; among these, 97 proteins showed a trend of downregulation and 9 proteins showed a trend of upregulation with increasing radiation dose. In addition, there were 21 and 8 proteins observed to have irregular trends with the 10 cGy irradiated group either having the highest or the lowest level among all three radiated doses. Moreover, two proteins, carboxypeptidase E and ubiquitin carboxyl-terminal hydrolase isozyme L1 were sensitive to ionizing radiation, but relatively independent of radiation dose. Conversely, proteasome activator complex subunit 2 protein appeared to be sensitive to the dose of radiation, as rapid upregulation of this protein was observed when radiation doses were increased from 3, to 10 or 200 cGy. These results suggest that different mechanisms of action exist at the secretome level for low and high doses of ionizing radiation.

  5. Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger.

    Science.gov (United States)

    Krijgsheld, Pauline; Nitsche, Benjamin M; Post, Harm; Levin, Ana M; Müller, Wally H; Heck, Albert J R; Ram, Arthur F J; Altelaar, A F Maarten; Wösten, Han A B

    2013-04-05

    Aspergillus niger is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain ΔflbA that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the ΔbrlA strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown ΔflbA colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of ΔflbA colonies. Of these, 18 proteins had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of flbA results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain ΔflbA has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve A. niger as a cell factory.

  6. Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

    Science.gov (United States)

    Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

    2015-08-04

    The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    Science.gov (United States)

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  8. Frustration-guided motion planning reveals conformational transitions in proteins.

    Science.gov (United States)

    Budday, Dominik; Fonseca, Rasmus; Leyendecker, Sigrid; van den Bedem, Henry

    2017-10-01

    Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics-inspired motion planning procedure called dCC-RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25 Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/. © 2017 Wiley Periodicals, Inc.

  9. The Penicillium echinulatum secretome on sugar cane bagasse.

    Directory of Open Access Journals (Sweden)

    Daniela A Ribeiro

    Full Text Available Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases. Glycoside hydrolase (GH family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.

  10. Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood Samples

    DEFF Research Database (Denmark)

    Boersema, P.J.; Geiger, T.; Wiśniewski, J.R.

    2013-01-01

    Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines....... In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting...

  11. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-08

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  12. Characterization of HSP27 phosphorylation sites in human atherosclerotic plaque secretome

    DEFF Research Database (Denmark)

    Durán, Mari-Carmen; Boeri-Erba, Elisabetta; Mohammed, Shabaz

    2007-01-01

    spectrometry (MS). Among the identified proteins, two isoforms of heat shock protein 27 (HSP27), a protein recently described as a potential biomarker of atherosclerosis, were detected. However, the putative mechanisms in which HSP27 isoforms could be involved in the atherosclerotic process are unknown. Thus......, the role that phosphorylated HSP27 could play in the atherosclerotic process is actually under study. The present work shows the strategies employed to characterize the phosphorylation in the HSP27 secreted by atheroma plaque samples. The application of liquid chromatography tandem mass spectrometry (MS......-lymphocytes). These interactions can be mediated by proteins secreted from these cells, which therefore exert an important role in the atherosclerotic process. We recently described a novel strategy for the characterization of the human atherosclerotic plaque secretome, combining two-dimensional gel electrophoresis and mass...

  13. Unraveling the in vitro secretome of the phytopathogen Botrytis cinerea to understand the interaction with its hosts

    Directory of Open Access Journals (Sweden)

    Raquel eGonzález-Fernández

    2015-10-01

    Full Text Available Botrytis cinerea is a necrotrophic fungus with high adaptability to different environments and hosts. It secretes a large number of extracellular proteins, which favor plant tissue penetration and colonization, thus contributing to virulence. Secretomics is a proteomics sub-discipline which study the secreted proteins and their secretion mechanisms, so-called secretome. By using proteomics as experimental approach, many secreted proteins by B. cinerea have been identified from in vitro experiments, and belonging to different functional categories: i cell wall-degrading enzymes such as pectinesterases, and endo-polygalacturonases; ii proteases involved in host protein degradation such as an aspartic protease; iii proteins related to the oxidative burst such as glyoxal oxidase; iv proteins which may induce the plant hypersensitive response such as a cerato-platanin domain-containing protein; and v proteins related to production and secretion of toxins such as malate dehydrogenase. In this mini-review, we made an overview of the proteomics contribution to the study and knowledge of the B. cinerea extracellular secreted proteins based on our current work carried out from in vitro experiments, and recent published papers both in vitro and in planta studies on this fungi. We hypothesize on the putative functions of these secreted proteins, and their connection to the biology of the B. cinerea interaction with its hosts.

  14. Human astrocytes: secretome profiles of cytokines and chemokines.

    Directory of Open Access Journals (Sweden)

    Sung S Choi

    Full Text Available Astrocytes play a key role in maintenance of neuronal functions in the central nervous system by producing various cytokines, chemokines, and growth factors, which act as a molecular coordinator of neuron-glia communication. At the site of neuroinflammation, astrocyte-derived cytokines and chemokines play both neuroprotective and neurotoxic roles in brain lesions of human neurological diseases. At present, the comprehensive profile of human astrocyte-derived cytokines and chemokines during inflammation remains to be fully characterized. We investigated the cytokine secretome profile of highly purified human astrocytes by using a protein microarray. Non-stimulated human astrocytes in culture expressed eight cytokines, including G-CSF, GM-CSF, GROα (CXCL1, IL-6, IL-8 (CXCL8, MCP-1 (CCL2, MIF and Serpin E1. Following stimulation with IL-1β and TNF-α, activated astrocytes newly produced IL-1β, IL-1ra, TNF-α, IP-10 (CXCL10, MIP-1α (CCL3 and RANTES (CCL5, in addition to the induction of sICAM-1 and complement component 5. Database search indicated that most of cytokines and chemokines produced by non-stimulated and activated astrocytes are direct targets of the transcription factor NF-kB. These results indicated that cultured human astrocytes express a distinct set of NF-kB-target cytokines and chemokines in resting and activated conditions, suggesting that the NF-kB signaling pathway differentially regulates gene expression of cytokines and chemokines in human astrocytes under physiological and inflammatory conditions.

  15. Proteomic techniques for characterisation of mesenchymal stem cell secretome.

    Czech Academy of Sciences Publication Activity Database

    Kupcová Skalníková, Helena

    2013-01-01

    Roč. 95, č. 12 (2013), s. 2196-2211 ISSN 0300-9084 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR TA01011466 Institutional support: RVO:67985904 Keywords : mesenchymal stem cells * secretome * exosome * conditioned medium * proteomics Subject RIV: CE - Biochemistry Impact factor: 3.123, year: 2013

  16. Eavesdropping on altered cell-to-cell signaling in cancer by secretome profiling.

    Science.gov (United States)

    Klinke, David J

    2016-01-01

    In the past decade, cumulative clinical experiences with molecular targeted therapies and immunotherapies for cancer have promoted a shift in our conceptual understanding of cancer. This view shifted from viewing solid tumors as a homogeneous mass of malignant cells to viewing tumors as heterogeneous structures that are dynamically shaped by intercellular interactions among the variety of stromal, immune, and malignant cells present within the tumor microenvironment. As in any dynamic system, identifying how cells communicate to maintain homeostasis and how this communication is altered during oncogenesis are key hurdles for developing therapies to restore normal tissue homeostasis. Here, I discuss tissues as dynamic systems, using the mammary gland as an example, and the evolutionary concepts applied to oncogenesis. Drawing from these concepts, I present 2 competing hypotheses for how intercellular communication might be altered during oncogenesis. As an initial test of these competing hypotheses, a recent secretome comparison between normal human mammary and HER2+ breast cancer cell lines suggested that the particular proteins secreted by the malignant cells reflect a convergent evolutionary path associated with oncogenesis in a specific anatomical niche, despite arising in different individuals. Overall, this study illustrates the emerging power of secretome proteomics to probe, in an unbiased way, how intercellular communication changes during oncogenesis.

  17. Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

    Science.gov (United States)

    Zeng, Tian; Holmer, Rens; Hontelez, Jan; Te Lintel-Hekkert, Bas; Marufu, Lucky; de Zeeuw, Thijs; Wu, Fangyuan; Schijlen, Elio; Bisseling, Ton; Limpens, Erik

    2018-05-01

    Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  18. Network based approaches reveal clustering in protein point patterns

    Science.gov (United States)

    Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

    2014-03-01

    Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

  19. Lignin depolymerization by fungal secretomes and a microbial sink

    Energy Technology Data Exchange (ETDEWEB)

    Salvachúa, Davinia; Katahira, Rui; Cleveland, Nicholas S.; Khanna, Payal; Resch, Michael G.; Black, Brenna A.; Purvine, Samuel O.; Zink, Erika M.; Prieto, Alicia; Martínez, María J.; Martínez, Angel T.; Simmons, Blake A.; Gladden, John M.; Beckham, Gregg T.

    2016-08-25

    In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high

  20. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    International Nuclear Information System (INIS)

    Kawahara, R.; Simabuco, F.M.; Yokoo, S.; Paes Leme, A.F.; Sherman, N.

    2012-01-01

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  1. N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Xianyu Li

    Full Text Available Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC cell lines with low (MHCC97L or high (HCCLM3 metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC, with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

  2. Cotranslational protein folding reveals the selective use of ...

    Indian Academy of Sciences (India)

    to fold properly by decelerating the translation rate at these sites. Thus the cotranslational protein folding is believed to be true for many proteins and is an important selection factor for the selective codon usage to optimize proper gene expres- sion and function (Komar 2009). A web server CS and S has been created by ...

  3. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. Copyright © 2015, American Association for the Advancement of Science.

  4. Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Jun Ni

    Full Text Available The Auxin/Indole-3-Acetic Acid (Aux/IAA and Auxin Response Factor (ARF are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S. The mutated OsARF(WSs can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WSs in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

  5. Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

    Directory of Open Access Journals (Sweden)

    Perry Evans

    Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

  6. Ribosomal history reveals origins of modern protein synthesis.

    Directory of Open Access Journals (Sweden)

    Ajith Harish

    Full Text Available The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17 and the oldest substructure (the ribosomal ratchet in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  7. Secretome Analysis Identifies Potential Pathogenicity/Virulence Factors of Tilletia indica, a Quarantined Fungal Pathogen Inciting Karnal Bunt Disease in Wheat.

    Science.gov (United States)

    Pandey, Vishakha; Singh, Manoj; Pandey, Dinesh; Marla, Soma; Kumar, Anil

    2018-04-01

    Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity/virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI-TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host-derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity/virulence factors homologs that were further subjected to sequence- and structure-based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity/virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

    Science.gov (United States)

    Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

    2014-10-01

    Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Genetic similarity of soybean genotypes revealed by seed protein

    Directory of Open Access Journals (Sweden)

    Nikolić Ana

    2005-01-01

    Full Text Available More accurate and complete descriptions of genotypes could help determinate future breeding strategies and facilitate introgression of new genotypes in current soybean genetic pool. The objective of this study was to characterize 20 soybean genotypes from the Maize Research Institute "Zemun Polje" collection, which have good agronomic performances, high yield, lodging and drought resistance, and low shuttering by seed proteins as biochemical markers. Seed proteins were isolated and separated by PAA electrophoresis. On the basis of the presence/absence of protein fractions coefficients of similarity were calculated as Dice and Roger and Tanamoto coefficient between pairs of genotypes. The similarity matrix was submitted for hierarchical cluster analysis of un weighted pair group using arithmetic average (UPGMA method and necessary computation were performed using NTSYS-pc program. Protein seed analysis confirmed low level of genetic diversity in soybean. The highest genetic similarity was between genotypes P9272 and Kador. According to obtained results, soybean genotypes were assigned in two larger groups and coefficients of similarity showed similar results. Because of the lack of pedigree data for analyzed genotypes, correspondence with marker data could not be determined. In plant with a narrow genetic base in their gene pool, such as soybean, protein markers may not be sufficient for characterization and study of genetic diversity.

  10. A comparative secretome analysis of industrial Aspergillus oryzae and its spontaneous mutant ZJGS-LZ-21.

    Science.gov (United States)

    Zhu, Yuanyuan; Liang, Xinle; Zhang, Hong; Feng, Wei; Liu, Ye; Zhang, Fuming; Linhardt, Robert J

    2017-05-02

    Aspergillus oryzae koji plays a crucial role in fermented food products due to the hydrolytic activities of secreted enzymes. In the present study, we performed a comparative secretome analysis of the industrial strain of Aspergillus oryzae 3.042 and its spontaneous mutantZJGS-LZ-21. One hundred and fifty two (152) differential protein spots were excised (p<0.05), and 25 proteins were identified. Of the identified proteins, 91.3% belonged to hydrolytic enzymes acting on carbohydrates or proteins. Consistent with their enzyme activities, the expression of 14 proteins involved in the degradation of cellulose, hemicellulose, starch and proteins, increased in the ZJGS-LZ-21isolate. In particular, increased levels of acid protease (Pep) may favor the degradation of soy proteins in acidic environments and promote the cleavage of allergenic soybean proteins in fermentation, resulting in improvements of product safety and quality. The ZJGS-LZ-21 isolate showed higher protein secretion and increased hydrolytic activities than did strain 3.042, indicating its promising application in soybean paste fermentation. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin.

    Science.gov (United States)

    A L Rocha, Vanessa; N Maeda, Roberto; Pereira, Nei; F Kern, Marcelo; Elias, Luisa; Simister, Rachael; Steele-King, Clare; Gómez, Leonardo D; McQueen-Mason, Simon J

    2016-03-01

    This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS-MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono-oxygenases (AA9), carbohydrate-binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two-fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327-336, 2016. © 2016 American Institute of Chemical Engineers.

  12. Two-photon polarization microscopy reveals protein structure and function

    Czech Academy of Sciences Publication Activity Database

    Lazar, Josef; Bondar, Alexey; Timr, S.; Firestein, S. J.

    2011-01-01

    Roč. 8, č. 8 (2011), s. 684-U120 ISSN 1548-7091 Institutional research plan: CEZ:AV0Z60870520 Keywords : green fluorescent proteins * living cells * in-vivo * indicators * anisotropy * activacion * dissociation * orientation * calmodulin * membranes Subject RIV: CE - Biochemistry Impact factor: 19.276, year: 2011

  13. Structure of Drosophila Oskar reveals a novel RNA binding protein

    Science.gov (United States)

    Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

    2015-01-01

    Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

  14. Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

    Science.gov (United States)

    Han, Dohyun; Jin, Jonghwa; Woo, Jongmin; Min, Hophil; Kim, Youngsoo

    2014-07-01

    Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole-cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte-neuron communication. To build a reference proteome, we established a C8-D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two-step digestion, filter-aided sample preparation, StageTip-based high pH fractionation, and high-resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High-confidence whole-cell proteomes and secretomes are valuable resources in studying astrocyte function by label-free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 (http://proteomecentral.proteomexchange.org/dataset/PXD000501). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Oxidation of Wine Polyphenols by Secretomes of Wild Botrytis cinerea Strains from White and Red Grape Varieties and Determination of Their Specific Laccase Activity.

    Science.gov (United States)

    Zimdars, Sabrina; Hitschler, Julia; Schieber, Andreas; Weber, Fabian

    2017-12-06

    Processing of Botrytis cinerea-infected grapes leads to enhanced enzymatic browning reactions mainly caused by the enzyme laccase which is able to oxidize a wide range of phenolic compounds. The extent of color deterioration depends on the activity of the enzymes secreted by the fungus. The present study revealed significant differences in the oxidative properties of secretomes of several B. cinerea strains isolated from five grape varieties. The presumed laccase-containing secretomes varied in their catalytic activity toward six phenolic compounds present in grapes. All strains led to identical product profiles for five of six substrates, but two strains showed deviating product profiles during gallic acid oxidation. Fast oxidation of caffeic acid, ferulic acid, and malvidin 3-O-glucoside was observed. Product formation rates and relative product concentrations were determined. The results reflect the wide range of enzyme activity and the corresponding different impact on color deterioration by B. cinerea.

  16. Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

    Science.gov (United States)

    Chowdhury, S Roy; Cao, Jin; He, Yufan; Lu, H Peter

    2018-03-27

    Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.

  17. High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation

    Science.gov (United States)

    Clutterbuck, Abigail L.; Smith, Julia R.; Allaway, David; Harris, Pat; Liddell, Susan; Mobasheri, Ali

    2011-01-01

    This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10 ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis. PMID:21354348

  18. Secretomic Insight into Glucose Metabolism of Aspergillus brasiliensis in Solid-State Fermentation.

    Science.gov (United States)

    Volke-Sepulveda, Tania; Salgado-Bautista, Daniel; Bergmann, Carl; Wells, Lance; Gutierrez-Sanchez, Gerardo; Favela-Torres, Ernesto

    2016-10-07

    The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO 2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.

  19. Secretome analysis defines the major role of SecDF in Staphylococcus aureus virulence.

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    Chantal Quiblier

    Full Text Available The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections.

  20. Growth-dependent secretome of Candida utilis

    NARCIS (Netherlands)

    Buerth, C.; Heilmann, C.J.; Klis, F.M.; de Koster, C.G.; Ernst, J.F.; Tielker, D.

    2011-01-01

    Recently, the food yeast Candida utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. utilis. Cells were cultivated to the exponential or

  1. Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

    NARCIS (Netherlands)

    Oliveira, L.; Paiva, P.B.; Paiva, A.C.; Vriend, G.

    2003-01-01

    Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it

  2. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

    Science.gov (United States)

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-09-27

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  3. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Je Min, E-mail: jemin@knu.ac.kr [Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul (Korea, Republic of); Department of Horticultural Science, Kyungpook National University, Daegu (Korea, Republic of); Lee, Sang-Jik [Biotechnology Institute, Nongwoo Bio Co, Ltd, Yeoju (Korea, Republic of); Department of Plant Biology, Cornell University, Ithaca, NY (United States); Rose, Jocelyn K.C. [Department of Plant Biology, Cornell University, Ithaca, NY (United States); Yeam, Inhwa [Department of Horticulture and Breeding, Andong National University, Andong (Korea, Republic of); Kim, Byung-Dong [Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul (Korea, Republic of)

    2014-04-18

    Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.

  4. Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

    International Nuclear Information System (INIS)

    Lee, Je Min; Lee, Sang-Jik; Rose, Jocelyn K.C.; Yeam, Inhwa; Kim, Byung-Dong

    2014-01-01

    Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening

  5. Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1 in Maize

    Directory of Open Access Journals (Sweden)

    Jin Yu

    2014-05-01

    Full Text Available Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L. and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1; which did not carry a gametophytic factor and W22 (Ga1, a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1 and W22 (Ga1. A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1 and W22 (Ga1 whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1 and W22 (Ga1. However, in pollen, 2 proteins were identified only in the W22 (ga1 and 12 proteins only in the W22 (Ga1 whereas 10 proteins were confirmed from the both of W22 (ga1 and W22 (Ga1. In contrary, 10 proteins were appeared only in the pistil of W22 (ga1 and 7 proteins from W22 (Ga1 while 3 proteins confirmed in the both of W22 (ga1 and W22 (Ga1. Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize.

  6. The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Ana O. Pires

    2014-01-01

    Full Text Available The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs isolated from human bone-marrow (BMSCs and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y. For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.

  7. Facile synthesis of novel magnetic silica nanoparticles functionalized with layer-by-layer detonation nanodiamonds for secretome study.

    Science.gov (United States)

    Li, Hong; Wang, Yi; Zhang, Lei; Lu, Haojie; Zhou, Zhongjun; Wei, Liming; Yang, Pengyuan

    2015-12-07

    Novel magnetic silica nanoparticles functionalized with layer-by-layer detonation nanodiamonds (dNDs) were prepared by coating single submicron-size magnetite particles with silica and subsequently modified with dNDs. The resulting layer-by-layer dND functionalized magnetic silica microspheres (Fe3O4@SiO2@[dND]n) exhibit a well-defined magnetite-core-silica-shell structure and possess a high content of magnetite, which endow them with high dispersibility and excellent magnetic responsibility. Meanwhile, dNDs are known for their high affinity and biocompatibility towards peptides or proteins. Thus, a novel convenient, fast and efficient pretreatment approach of low-abundance peptides or proteins was successfully established with Fe3O4@SiO2@[dND]n microspheres. The signal intensity of low-abundance peptides was improved by at least two to three orders of magnitude in mass spectrometry analysis. The novel microsphere also showed good tolerance to salt. Even with a high concentration of salt, peptides or proteins could be isolated effectively from samples. Therefore, the convenient and efficient enrichment process of this novel layer-by-layer dND-functionalized microsphere makes it a promising candidate for isolation of protein in a large volume of culture supernatant for secretome analysis. In the application of Fe3O4@SiO2@[dND]n in the secretome of hepatoma cells, 1473 proteins were identified and covered a broad range of pI and molecular weight, including 377 low molecular weight proteins.

  8. Energy landscape of all-atom protein-protein interactions revealed by multiscale enhanced sampling.

    Directory of Open Access Journals (Sweden)

    Kei Moritsugu

    2014-10-01

    Full Text Available Protein-protein interactions are regulated by a subtle balance of complicated atomic interactions and solvation at the interface. To understand such an elusive phenomenon, it is necessary to thoroughly survey the large configurational space from the stable complex structure to the dissociated states using the all-atom model in explicit solvent and to delineate the energy landscape of protein-protein interactions. In this study, we carried out a multiscale enhanced sampling (MSES simulation of the formation of a barnase-barstar complex, which is a protein complex characterized by an extraordinary tight and fast binding, to determine the energy landscape of atomistic protein-protein interactions. The MSES adopts a multicopy and multiscale scheme to enable for the enhanced sampling of the all-atom model of large proteins including explicit solvent. During the 100-ns MSES simulation of the barnase-barstar system, we observed the association-dissociation processes of the atomistic protein complex in solution several times, which contained not only the native complex structure but also fully non-native configurations. The sampled distributions suggest that a large variety of non-native states went downhill to the stable complex structure, like a fast folding on a funnel-like potential. This funnel landscape is attributed to dominant configurations in the early stage of the association process characterized by near-native orientations, which will accelerate the native inter-molecular interactions. These configurations are guided mostly by the shape complementarity between barnase and barstar, and lead to the fast formation of the final complex structure along the downhill energy landscape.

  9. Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment.

    Science.gov (United States)

    Salvachúa, Davinia; Martínez, Angel T; Tien, Ming; López-Lucendo, María F; García, Francisco; de Los Ríos, Vivian; Martínez, María Jesús; Prieto, Alicia

    2013-08-10

    Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases. In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases. The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

  10. The stem cell secretome and its role in brain repair.

    Science.gov (United States)

    Drago, Denise; Cossetti, Chiara; Iraci, Nunzio; Gaude, Edoardo; Musco, Giovanna; Bachi, Angela; Pluchino, Stefano

    2013-12-01

    Compelling evidence exists that non-haematopoietic stem cells, including mesenchymal (MSCs) and neural/progenitor stem cells (NPCs), exert a substantial beneficial and therapeutic effect after transplantation in experimental central nervous system (CNS) disease models through the secretion of immune modulatory or neurotrophic paracrine factors. This paracrine hypothesis has inspired an alternative outlook on the use of stem cells in regenerative neurology. In this paradigm, significant repair of the injured brain may be achieved by injecting the biologics secreted by stem cells (secretome), rather than implanting stem cells themselves for direct cell replacement. The stem cell secretome (SCS) includes cytokines, chemokines and growth factors, and has gained increasing attention in recent years because of its multiple implications for the repair, restoration or regeneration of injured tissues. Thanks to recent improvements in SCS profiling and manipulation, investigators are now inspired to harness the SCS as a novel alternative therapeutic option that might ensure more efficient outcomes than current stem cell-based therapies for CNS repair. This review discusses the most recent identification of MSC- and NPC-secreted factors, including those that are trafficked within extracellular membrane vesicles (EVs), and reflects on their potential effects on brain repair. It also examines some of the most convincing advances in molecular profiling that have enabled mapping of the SCS. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  11. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins

    Science.gov (United States)

    Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela

    2015-01-01

    Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950

  12. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

    Science.gov (United States)

    Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

    2016-04-15

    The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Rhabdovirus matrix protein structures reveal a novel mode of self-association.

    Directory of Open Access Journals (Sweden)

    Stephen C Graham

    2008-12-01

    Full Text Available The matrix (M proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus and from Lagos bat virus (genus: Lyssavirus, revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

  14. Major surfome and secretome profile of Streptococcus agalactiae from Nile tilapia (Oreochromis niloticus): Insight into vaccine development.

    Science.gov (United States)

    Li, Wei; Wang, Hai-Qing; He, Run-Zhen; Li, Yan-Wei; Su, You-Lu; Li, An-Xing

    2016-08-01

    Streptococcus agalactiae is a major piscine pathogen that is responsible for huge economic losses to the aquaculture industry. Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against S. agalactiae. The proteins of S. agalactiae exposed to the environment, including surface proteins and secretory proteins, are important targets for the immune system and they are likely to be good vaccine candidates. To obtain a precise profile of its surface proteins, S. agalactiae strain THN0901, which was isolated from tilapia (Oreochromis niloticus), was treated with proteinase K to cleave surface-exposed proteins, which were identified by liquid chromatography-tandem spectrometry (LC-MS/MS). Forty surface-associated proteins were identified, including ten proteins containing cell wall-anchoring motifs, eight lipoproteins, eleven membrane proteins, seven secretory proteins, three cytoplasmic proteins, and one unknown protein. In addition, culture supernatant proteins of S. agalactiae were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all of the Coomassie-stained bands were subsequently identified by LC-MS/MS. A total of twenty-six extracellular proteins were identified, including eleven secretory proteins, seven cell wall proteins, three membrane proteins, two cytoplasmic proteins and three unknown proteins. Of these, six highly expressed surface-associated and secretory proteins are putative to be vaccine candidate of piscine S. agalactiae. Moreover, immunogenic secreted protein, a highly expressed protein screened from the secretome in the present study, was demonstrated to induce high antibody titer in tilapia, and it conferred protection against S. agalactiae, as evidenced by the relative percent survival (RPS) 48.61± 8.45%. The data reported here narrow the scope of screening protective antigens, and provide guidance in the development of a novel

  15. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    Science.gov (United States)

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  16. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    Directory of Open Access Journals (Sweden)

    Robert Kalescky

    2016-04-01

    Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  17. Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

    Directory of Open Access Journals (Sweden)

    Yongbin Dong

    Full Text Available The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.

  18. Peptidomics and Secretomics of the Mammalian Peripheral Sensory-Motor System

    Science.gov (United States)

    Tillmaand, Emily G.; Yang, Ning; Kindt, Callie A. C.; Romanova, Elena V.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2015-12-01

    The dorsal root ganglion (DRG) and its anatomically and functionally associated spinal nerve and ventral and dorsal roots are important components of the peripheral sensory-motor system in mammals. The cells within these structures use a number of peptides as intercellular signaling molecules. We performed a variety of mass spectrometry (MS)-based characterizations of peptides contained within and secreted from these structures, and from isolated and cultured DRG cells. Liquid chromatography-Fourier transform MS was utilized in DRG and nerve peptidome analysis. In total, 2724 peptides from 296 proteins were identified in tissue extracts. Neuropeptides are among those detected, including calcitonin gene-related peptide I, little SAAS, and known hemoglobin-derived peptides. Solid phase extraction combined with direct matrix-assisted laser desorption/ionization time-of-flight MS was employed to investigate the secretome of these structures. A number of peptides were detected in the releasate from semi-intact preparations of DRGs and associated nerves, including neurofilament- and myelin basic protein-related peptides. A smaller set of analytes was observed in releasates from cultured DRG neurons. The peptide signals observed in the releasates have been mass-matched to those characterized and identified in homogenates of entire DRGs and associated nerves. This data aids our understanding of the chemical composition of the mammalian peripheral sensory-motor system, which is involved in key physiological functions such as nociception, thermoreception, itch sensation, and proprioception.

  19. Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

    Science.gov (United States)

    Sinha, Sudhir; Kosalai, K; Arora, Shalini; Namane, Abdelkader; Sharma, Pawan; Gaikwad, Anil N; Brodin, Priscille; Cole, Stewart T

    2005-07-01

    Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute

  20. Quantitative analysis of secretome from adipocytes regulated by insulin

    Institute of Scientific and Technical Information of China (English)

    Hu Zhou; Yuanyuan Xiao; Rongxia Li; Shangyu Hong; Sujun Li; Lianshui Wang; Rong Zeng; Kan Liao

    2009-01-01

    Adipocyte is not only a central player involved in storage and release of energy, but also in regulation of energy metabolism in other organs via secretion of pep-tides and proteins. During the pathogenesis of insulin resistance and type 2 diabetes, adipocytes are subjected to the increased levels of insulin, which may have a major impact on the secretion of adipokines. We have undertaken cleavable isotope-coded affinity tag (clCAT) and label-free quantitation approaches to identify and quantify secretory factors that are differen-tially secreted by 3T3-LI adipocytes with or without insulin treatment. Combination of clCAT and label-free results, there are 317 proteins predicted or annotated as secretory proteins. Among these secretory proteins, 179 proteins and 53 proteins were significantly up-regulated and down-regulated, respectively. A total of 77 reported adipokines were quantified in our study, such as adiponectin, cathepsin D, cystatin C, resistin, and transferrin. Western blot analysis of these adipo-kines confirmed the quantitative results from mass spectrometry, and revealed individualized secreting pat-terns of these proteins by increasing insulin dose. In addition, 240 proteins were newly identified and quanti-fied as secreted proteins from 3T3-L1 adipocytes in our study, most of which were up-regulated upon insulin treatment. Further comprehensive bioinformatics analysis revealed that the secretory proteins in extra-cellular matrix-receptor interaction pathway and glycan structure degradation pathway were significantly up-regulated by insulin stimulation.

  1. Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

    Science.gov (United States)

    Florencio, Camila; Cunha, Fernanda M; Badino, Alberto C; Farinas, Cristiane S; Ximenes, Eduardo; Ladisch, Michael R

    2016-08-01

    Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme

  2. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

    Science.gov (United States)

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-29

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

  3. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans.

    Science.gov (United States)

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C G; Benavente, Ricardo

    2012-10-09

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.

  4. Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

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    Samuel Hertig

    2016-06-01

    Full Text Available Molecular dynamics (MD simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery-the fact that the two sites involved influence one another in a symmetrical manner-can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

  5. Proteomic analysis reveals the diversity and complexity of membrane proteins in chickpea (Cicer arietinum L.

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    Jaiswal Dinesh Kumar

    2012-10-01

    Full Text Available Abstract Background Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. Results To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. Conclusions The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.

  6. Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

    Science.gov (United States)

    Yuan, Peijia; D'Lima, Nadia G; Slavoff, Sarah A

    2018-01-09

    Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

  7. Proteomics investigation reveals cell death-associated proteins of basidiomycete fungus Trametes versicolor treated with Ferruginol.

    Science.gov (United States)

    Chen, Yu-Han; Yeh, Ting-Feng; Chu, Fang-Hua; Hsu, Fu-Lan; Chang, Shang-Tzen

    2015-01-14

    Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton β-tubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)–multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi.

  8. Transcription Factor Repertoire of Necrotrophic Fungal Phytopathogen Ascochyta rabiei: Predominance of MYB Transcription Factors As Potential Regulators of Secretome

    Directory of Open Access Journals (Sweden)

    Sandhya Verma

    2017-06-01

    Full Text Available Transcription factors (TFs are the key players in gene expression and their study is highly significant for shedding light on the molecular mechanisms and evolutionary history of organisms. During host–pathogen interaction, extensive reprogramming of gene expression facilitated by TFs is likely to occur in both host and pathogen. To date, the knowledge about TF repertoire in filamentous fungi is in infancy. The necrotrophic fungus Ascochyta rabiei, that causes destructive Ascochyta blight (AB disease of chickpea (Cicer arietinum, demands more comprehensive study for better understanding of Ascochyta-legume pathosystem. In the present study, we performed the genome-wide identification and analysis of TFs in A. rabiei. Taking advantage of A. rabiei genome sequence, we used a bioinformatic approach to predict the TF repertoire of A. rabiei. For identification and classification of A. rabiei TFs, we designed a comprehensive pipeline using a combination of BLAST and InterProScan software. A total of 381 A. rabiei TFs were predicted and divided into 32 fungal specific families of TFs. The gene structure, domain organization and phylogenetic analysis of abundant families of A. rabiei TFs were also carried out. Comparative study of A. rabiei TFs with that of other necrotrophic, biotrophic, hemibiotrophic, symbiotic, and saprotrophic fungi was performed. It suggested presence of both conserved as well as unique features among them. Moreover, cis-acting elements on promoter sequences of earlier predicted A. rabiei secretome were also identified. With the help of published A. rabiei transcriptome data, the differential expression of TF and secretory protein coding genes was analyzed. Furthermore, comprehensive expression analysis of few selected A. rabiei TFs using quantitative real-time polymerase chain reaction revealed variety of expression patterns during host colonization. These genes were expressed in at least one of the time points tested post

  9. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

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    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

  10. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús

    2011-01-01

    Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one...... events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated...... proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized...

  11. Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers.

    Science.gov (United States)

    Rai, Rohit; Kaur, Baljit; Singh, Surender; Di Falco, Macros; Tsang, Adrian; Chadha, B S

    2016-09-01

    Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Hydrogen bonds of DsrD protein revealed by neutron crystallography

    International Nuclear Information System (INIS)

    Chatake, Toshiyuki; Higuchi, Yoshiki; Mizuno, Nobuhiro; Tanaka, Ichiro; Niimura, Nobuo; Morimoto, Yukio

    2008-01-01

    Hydrogen bonds of DNA-binding protein DsrD have been determined by neutron diffraction. In terms of proton donors and acceptors, DsrD protein shows striking differences from other proteins. The features of hydrogen bonds in DsrD protein from sulfate-reducing bacteria have been investigated by neutron protein crystallography. The function of DsrD has not yet been elucidated clearly, but its X-ray crystal structure revealed that it comprises a winged-helix motif and shows the highest structural homology to the DNA-binding proteins. Since any neutron structure of a DNA recognition protein has not yet been obtained, here detailed information on the hydrogen bonds in the winged-helix-motif protein is given and the following features found. (i) The number of hydrogen bonds per amino acid of DsrD is relatively fewer than for other proteins for which neutron structures were determined previously. (ii) Hydrogen bonds are localized between main-chain and main-chain atoms; there are few hydrogen bonds between main-chain and side-chain atoms and between side-chain and side-chain atoms. (iii) Hydrogen bonds inducted by protonation of specific amino acid residues (Glu50) seem to play an essential role in the dimerization of DsrD. The former two points are related to the function of the DNA-binding protein; the three-dimensional structure was mainly constructed by hydrogen bonds in main chains, while the side chains appeared to be used for another role. The latter point would be expected to contribute to the crystal growth of DsrD

  13. Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

    Science.gov (United States)

    Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

    2017-01-01

    Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

  14. Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry.

    Science.gov (United States)

    Asara, John M; Schweitzer, Mary H; Freimark, Lisa M; Phillips, Matthew; Cantley, Lewis C

    2007-04-13

    Fossilized bones from extinct taxa harbor the potential for obtaining protein or DNA sequences that could reveal evolutionary links to extant species. We used mass spectrometry to obtain protein sequences from bones of a 160,000- to 600,000-year-old extinct mastodon (Mammut americanum) and a 68-million-year-old dinosaur (Tyrannosaurus rex). The presence of T. rex sequences indicates that their peptide bonds were remarkably stable. Mass spectrometry can thus be used to determine unique sequences from ancient organisms from peptide fragmentation patterns, a valuable tool to study the evolution and adaptation of ancient taxa from which genomic sequences are unlikely to be obtained.

  15. Protein profiling in serum after traumatic brain injury in rats reveals potential injury markers.

    Science.gov (United States)

    Thelin, Eric Peter; Just, David; Frostell, Arvid; Häggmark-Månberg, Anna; Risling, Mårten; Svensson, Mikael; Nilsson, Peter; Bellander, Bo-Michael

    2018-03-15

    The serum proteome following traumatic brain injury (TBI) could provide information for outcome prediction and injury monitoring. The aim with this affinity proteomic study was to identify serum proteins over time and between normoxic and hypoxic conditions in focal TBI. Sprague Dawley rats (n=73) received a 3mm deep controlled cortical impact ("severe injury"). Following injury, the rats inhaled either a normoxic (22% O 2 ) or hypoxic (11% O 2 ) air mixture for 30min before resuscitation. The rats were sacrificed at day 1, 3, 7, 14 and 28 after trauma. A total of 204 antibodies targeting 143 unique proteins of interest in TBI research, were selected. The sample proteome was analyzed in a suspension bead array set-up. Comparative statistics and factor analysis were used to detect differences as well as variance in the data. We found that complement factor 9 (C9), complement factor B (CFB) and aldolase c (ALDOC) were detected at higher levels the first days after trauma. In contrast, hypoxia inducing factor (HIF)1α, amyloid precursor protein (APP) and WBSCR17 increased over the subsequent weeks. S100A9 levels were higher in hypoxic-compared to normoxic rats, together with a majority of the analyzed proteins, albeit few reached statistical significance. The principal component analysis revealed a variance in the data, highlighting clusters of proteins. Protein profiling of serum following TBI using an antibody based microarray revealed temporal changes of several proteins over an extended period of up to four weeks. Further studies are warranted to confirm our findings. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  16. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

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    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  17. Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics

    Directory of Open Access Journals (Sweden)

    Jörg eSchumacher

    2014-07-01

    Full Text Available The plant pathogen Pseudomonas syringae pv. tomato (DC3000 causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS. In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp. Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant’s capacity to recognise both pathogen and microbial determinants (PAMP/MAMP-triggered immunity. We have developed and employed MS-based shotgun and targeted proteomics to (i elucidate the extracellular and secretome composition of DC3000 and (ii evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopK1, HrpK1, HrpA1 and Avrpto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS to quantify HrpA1 and Avrpto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid Avrpto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.

  18. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Directory of Open Access Journals (Sweden)

    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  19. Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

    2012-01-01

    Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

  20. Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans

    OpenAIRE

    Fraune, Johanna; Alsheimer, Manfred; Volff, Jean-Nicolas; Busch, Karoline; Fraune, Sebastian; Bosch, Thomas C. G.; Benavente, Ricardo

    2012-01-01

    The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed...

  1. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Proteomic identification of secreted proteins of Propionibacterium acnes

    Directory of Open Access Journals (Sweden)

    Holland Carsten

    2010-08-01

    Full Text Available Abstract Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS. A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence

  3. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

    2013-01-01

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

    Science.gov (United States)

    De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

    2016-02-01

    Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

    Science.gov (United States)

    Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

    2016-11-23

    Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

  7. Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

    Science.gov (United States)

    Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

    2011-10-01

    The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

  8. Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential

    DEFF Research Database (Denmark)

    Sultan, Abida; Frisvad, Jens Christian; Andersen, Birgit

    2017-01-01

    The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface......-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown...... on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases...

  9. Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

    Science.gov (United States)

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2013-06-07

    Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

  10. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

    Directory of Open Access Journals (Sweden)

    Sibley L David

    2005-12-01

    Full Text Available Abstract Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex, and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery. In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility.

  11. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus.

    Directory of Open Access Journals (Sweden)

    Mingwei Huang

    2015-08-01

    Full Text Available Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.

  12. The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

    International Nuclear Information System (INIS)

    Das, Debanu; Finn, Robert D.; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB. Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA-OmlA proteins and hence are likely to function as inhibitory proteins

  13. Interplay of protein and DNA structure revealed in simulations of the lac operon.

    Directory of Open Access Journals (Sweden)

    Luke Czapla

    Full Text Available The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

  14. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    Science.gov (United States)

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Interplay of protein and DNA structure revealed in simulations of the lac operon.

    Science.gov (United States)

    Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

    2013-01-01

    The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

  16. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    Science.gov (United States)

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

    Directory of Open Access Journals (Sweden)

    Dimitar V Pachov

    2015-07-01

    Full Text Available Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key

  18. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  19. Proteomic investigation of aphid honeydew reveals an unexpected diversity of proteins.

    Directory of Open Access Journals (Sweden)

    Ahmed Sabri

    Full Text Available Aphids feed on the phloem sap of plants, and are the most common honeydew-producing insects. While aphid honeydew is primarily considered to comprise sugars and amino acids, its protein diversity has yet to be documented. Here, we report on the investigation of the honeydew proteome from the pea aphid Acyrthosiphon pisum. Using a two-Dimensional Differential in-Gel Electrophoresis (2D-Dige approach, more than 140 spots were isolated, demonstrating that aphid honeydew also represents a diverse source of proteins. About 66% of the isolated spots were identified through mass spectrometry analysis, revealing that the protein diversity of aphid honeydew originates from several organisms (i.e. the host aphid and its microbiota, including endosymbiotic bacteria and gut flora. Interestingly, our experiments also allowed to identify some proteins like chaperonin, GroEL and Dnak chaperones, elongation factor Tu (EF-Tu, and flagellin that might act as mediators in the plant-aphid interaction. In addition to providing the first aphid honeydew proteome analysis, we propose to reconsider the importance of this substance, mainly acknowledged to be a waste product, from the aphid ecology perspective.

  20. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

  1. Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

    Science.gov (United States)

    Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

    2005-01-01

    CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

  2. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  3. Therapeutic Potential of Dental Pulp Stem Cell Secretome for Alzheimer’s Disease Treatment: An In Vitro Study

    Directory of Open Access Journals (Sweden)

    Nermeen El-Moataz Bellah Ahmed

    2016-01-01

    Full Text Available The secretome obtained from stem cell cultures contains an array of neurotrophic factors and cytokines that might have the potential to treat neurodegenerative conditions. Alzheimer’s disease (AD is one of the most common human late onset and sporadic neurodegenerative disorders. Here, we investigated the therapeutic potential of secretome derived from dental pulp stem cells (DPSCs to reduce cytotoxicity and apoptosis caused by amyloid beta (Aβ peptide. We determined whether DPSCs can secrete the Aβ-degrading enzyme, neprilysin (NEP, and evaluated the effects of NEP expression in vitro by quantitating Aβ-degrading activity. The results showed that DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1, and GM-CSF compared to those of bone marrow and adipose stem cells. Moreover, treatment with DPSC secretome significantly decreased the cytotoxicity of Aβ peptide by increasing cell viability compared to nontreated cells. In addition, DPSC secretome stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. Furthermore, neprilysin enzyme was detected in DPSC secretome and succeeded in degrading Aβ1–42 in vitro in 12 hours. In conclusion, our study demonstrates that DPSCs may serve as a promising source for secretome-based treatment of Alzheimer’s disease.

  4. Integration of Breast Cancer Secretomes with Clinical Data Elucidates Potential Serum Markers for Disease Detection, Diagnosis, and Prognosis.

    Science.gov (United States)

    Ziegler, Yvonne S; Moresco, James J; Yates, John R; Nardulli, Ann M

    2016-01-01

    Cancer cells secrete factors that influence adjacent cell behavior and can lead to enhanced proliferation and metastasis. To better understand the role of these factors in oncogenesis and disease progression, estrogen and progesterone receptor positive MCF-7 cells, triple negative breast cancer MDA-MB-231, DT22, and DT28 cells, and MCF-10A non-transformed mammary epithelial cells were grown in 3D cultures. A special emphasis was placed on triple negative breast cancer since these tumors are highly aggressive and no targeted treatments are currently available. The breast cancer cells secreted factors of variable potency that stimulated proliferation of the relatively quiescent MCF-10A cells. The conditioned medium from each cell line was subjected to mass spectrometry analysis and a variety of secreted proteins were identified including glycolytic enzymes, proteases, protease inhibitors, extracellular matrix proteins, and insulin-like growth factor binding proteins. An investigation of the secretome from each cell line yielded clues about strategies used for breast cancer proliferation and metastasis. Some of the proteins we identified may be useful in the development of a serum-based test for breast cancer detection, diagnosis, prognosis, and monitoring.

  5. REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

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    Hao Yang

    2018-01-01

    Full Text Available Bryostatin 1 (henceforth bryostatin is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

  6. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

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    Martin Löwer

    Full Text Available Exported proteases of Helicobacter pylori (H. pylori are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI. Among these, we found the predicted serine protease HtrA (Hp1019, which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

  7. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

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    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  8. Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

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    Dennis J Templeton

    2010-11-01

    Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

  9. Investigating the secretome : Lessons about the cells that comprise the heart

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Van Eyk, J.E.

    2012-01-01

    Roč. 5, č. 1 (2012), o8-o18 ISSN 1942-325X Institutional research plan: CEZ:AV0Z40310501 Keywords : secretomes * proteomics * cardiovascular diseases Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 6.728, year: 2012

  10. Comparative transcriptome and secretome analysis of wood decay fungi Postia placenta and Phanerochaete chrysosporium

    Science.gov (United States)

    Amber J. Vanden Wymelenberg; Jill Gaskell; Michael Mozuch; Grzegorz Sabat; John Ralph; Oleksandr Skyba; Shawn D Mansfield; Robert A. Blanchette; Diego Martinez; Igor Grigoriev; Philip J Kersten; Daniel Cullen

    2010-01-01

    Cellulose degradation by brown rot fungi, such as Postia placenta, is poorly understood relative to the phylogenetically related white rot basidiomycete, Phanerochaete chrysosporium. To elucidate the number, structure, and regulation of genes involved in lignocellulosic cell wall attack, secretome and transcriptome analyses were performed on both wood decay fungi...

  11. Skeletal muscle secretome in Duchenne muscular dystrophy: a pivotal anti-inflammatory role of adiponectin.

    Science.gov (United States)

    Lecompte, S; Abou-Samra, M; Boursereau, R; Noel, L; Brichard, S M

    2017-07-01

    Persistent inflammation exacerbates the progression of Duchenne muscular dystrophy (DMD). The hormone, adiponectin (ApN), which is decreased in the metabolic syndrome, exhibits anti-inflammatory properties on skeletal muscle and alleviates the dystrophic phenotype of mdx mice. Here, we investigate whether ApN retains its anti-inflammatory action in myotubes obtained from DMD patients. We unravel the underlying mechanisms by studying the secretome and the early events of ApN. Primary cultures of myotubes from DMD and control patients were treated or not by ApN after an inflammatory challenge. Myokines secreted in medium were identified by cytokine antibody-arrays and ELISAs. The early events of ApN signaling were assessed by abrogating selected genes. ApN retained its anti-inflammatory properties in both dystrophic and control myotubes. Profiling of secretory products revealed that ApN downregulated the secretion of two pro-inflammatory factors (TNFα and IL-17A), one soluble receptor (sTNFRII), and one chemokine (CCL28) in DMD myotubes, while upregulating IL-6 that exerts some anti-inflammatory effects. These changes were explained by pretranslational mechanisms. Earlier events of the ApN cascade involved AdipoR1, the main receptor for muscle, and the AMPK-SIRT1-PGC-1α axis leading, besides alteration of the myokine profile, to the upregulation of utrophin A (a dystrophin analog). ApN retains its beneficial properties in dystrophic muscles by activating the AdipoR1-AMPK-SIRT1-PGC-1α pathway, thereby inducing a shift in the secretion of downstream myokines toward a less inflammatory profile while upregulating utrophin. ApN, the early events of the cascade and downstream myokines may be therapeutic targets for the management of DMD.

  12. Tree ferns: monophyletic groups and their relationships as revealed by four protein-coding plastid loci.

    Science.gov (United States)

    Korall, Petra; Pryer, Kathleen M; Metzgar, Jordan S; Schneider, Harald; Conant, David S

    2006-06-01

    Tree ferns are a well-established clade within leptosporangiate ferns. Most of the 600 species (in seven families and 13 genera) are arborescent, but considerable morphological variability exists, spanning the giant scaly tree ferns (Cyatheaceae), the low, erect plants (Plagiogyriaceae), and the diminutive endemics of the Guayana Highlands (Hymenophyllopsidaceae). In this study, we investigate phylogenetic relationships within tree ferns based on analyses of four protein-coding, plastid loci (atpA, atpB, rbcL, and rps4). Our results reveal four well-supported clades, with genera of Dicksoniaceae (sensu ) interspersed among them: (A) (Loxomataceae, (Culcita, Plagiogyriaceae)), (B) (Calochlaena, (Dicksonia, Lophosoriaceae)), (C) Cibotium, and (D) Cyatheaceae, with Hymenophyllopsidaceae nested within. How these four groups are related to one other, to Thyrsopteris, or to Metaxyaceae is weakly supported. Our results show that Dicksoniaceae and Cyatheaceae, as currently recognised, are not monophyletic and new circumscriptions for these families are needed.

  13. Proteomic analysis reveals changes in carbohydrate and protein metabolism associated with broiler breast myopathy.

    Science.gov (United States)

    Kuttappan, Vivek A; Bottje, Walter; Ramnathan, Ranjith; Hartson, Steven D; Coon, Craig N; Kong, Byung-Whi; Owens, Casey M; Vazquez-Añon, Mercedes; Hargis, Billy M

    2017-08-01

    White Striping (WS) and Woody Breast (WB) are 2 conditions that adversely affect consumer acceptance as well as quality of poultry meat and meat products. Both WS and WB are characterized with degenerative myopathic changes. Previous studies showed that WS and WB in broiler fillets could result in higher ultimate pH, increased drip loss, and decreased marinade uptake. The main objective of the present study was to compare the proteomic profiles of muscle tissue (n = 5 per group) with either NORM (no or few minor myopathic lesions) or SEV (with severe myopathic changes). Proteins were extracted from these samples and analyzed using a hybrid LTQ-OrbitrapXL mass spectrometer (LC-MS/MS). Over 800 proteins were identified in the muscle samples, among which 141 demonstrated differential (P < 0.05) expression between NORM and SEV. The set of differentially (P < 0.05) expressed proteins was uploaded to Ingenuity Pathway Analysis® (IPA) software to determine the associated biological networks and pathways. The IPA analysis showed that eukaryotic initiation factor-2 (eIF-2) signaling, mechanistic target of rapamycin (mTOR) signaling, as well as regulation of eIF4 and p70S6K signaling were the major canonical pathways up-regulated (P < 0.05) in SEV muscle compared to NORM. The up-regulation of these pathways indicate an increase in protein synthesis which could be part of the rapid growth as well as cellular stress associated with ongoing muscle degeneration and the attempt to repair tissue damage in SEV birds. Furthermore, IPA analysis revealed that glycolysis and gluconeogenesis were the major down-regulated (P < 0.05) canonical pathways in SEV with respect to NORM muscle. Down-regulation of these pathways could be the reason for higher ultimate pH seen in SEV muscle samples indicating reduced glycolytic potential. In conclusion, comparison of proteomic profiles of NORM and SEV muscle samples showed differences in protein profile which explains some of the observed

  14. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.

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    Rahul Navale

    Full Text Available Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8 employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64 and aspartic (pepstatin protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine, indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in

  15. The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

    Science.gov (United States)

    Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

    2017-12-01

    Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

  16. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

    Science.gov (United States)

    Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

    2015-01-01

    Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

  17. Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture.

    Science.gov (United States)

    Eswaran, Jeyanthy; Bernad, Antonio; Ligos, Jose M; Guinea, Barbara; Debreczeni, Judit E; Sobott, Frank; Parker, Sirlester A; Najmanovich, Rafael; Turk, Benjamin E; Knapp, Stefan

    2008-01-01

    The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

  18. Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Berle Magnus

    2011-05-01

    Full Text Available Abstract Background The mechanisms behind formation and filling of intracranial arachnoid cysts (AC are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1 Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2 Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF and plasma. Methods AC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM initiated detection and sequence analysis. Results We did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC. In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that

  19. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation.

    Science.gov (United States)

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C; Farese, Robert V

    2014-07-01

    Accurate protein inventories are essential for understanding an organelle's functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  20. Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

    Science.gov (United States)

    Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

    2007-01-01

    Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

  1. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

    Directory of Open Access Journals (Sweden)

    Alessandro Pandini

    Full Text Available Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM domains (amino-terminal (FliGN, middle (FliGM and FliGC as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6. FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM

  2. An Interactome-Centered Protein Discovery Approach Reveals Novel Components Involved in Mitosome Function and Homeostasis in Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Samuel Rout

    2016-12-01

    Full Text Available Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30-40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein

  3. Postnatal development of cerebellar zones revealed by neurofilament heavy chain protein expression

    Directory of Open Access Journals (Sweden)

    Joshua J White

    2013-05-01

    Full Text Available The cerebellum is organized into parasagittal zones that control sensory-motor behavior. Although the architecture of adult zones is well understood, very little is known about how zones emerge during development. Understanding the process of zone formation is an essential step towards unraveling how circuits are constructed to support specific behaviors. Therefore, we focused this study on postnatal development to determine the spatial and temporal changes that establish zonal patterns during circuit formation. We used a combination of wholemount and tissue section immunohistochemistry in mice to show that the cytoskeletal protein neurofilament heavy chain (NFH is a robust marker for postnatal cerebellar zonal patterning. The patterned expression of NFH is initiated shortly after birth, and compared to the domains of several known zonal markers such as zebrin II, HSP25, neurogranin, and phospholipase Cβ4 (PLCβ4, NFH does not exhibit transient expression patterns that are typically remodeled between stages, and the adult zones do not emerge after a period of uniform expression in all lobules. Instead, we found that throughout postnatal development NFH gradually reveals distinct zones in each cerebellar lobule. The boundaries of individual NFH zones sharpen over time, as zones are refined during the second and third weeks after birth. Double labeling with neurogranin and PLCβ4 further revealed that although the postnatal expression of NFH is spatially and temporally unique, its pattern of zones respects a fundamental and well-known molecular topography in the cerebellum. The dynamics of NFH expression support the hypothesis that adult circuits are derived from an embryonic map that is refined into zones during the first three-weeks of life.

  4. Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli

    Science.gov (United States)

    Johnson, Brant R.; Hymes, Jeffrey; Sanozky-Dawes, Rosemary; Henriksen, Emily DeCrescenzo

    2015-01-01

    The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa. PMID:26475115

  5. Stem cell secretome-rich nanoclay hydrogel: a dual action therapy for cardiovascular regeneration

    Science.gov (United States)

    Waters, Renae; Pacelli, Settimio; Maloney, Ryan; Medhi, Indrani; Ahmed, Rafeeq P. H.; Paul, Arghya

    2016-03-01

    A nanocomposite hydrogel with photocrosslinkable micro-porous networks and a nanoclay component was successfully prepared to control the release of growth factor-rich stem cell secretome. The proven pro-angiogenic and cardioprotective potential of this new bioactive system provides a valuable therapeutic platform for cardiac tissue repair and regeneration.A nanocomposite hydrogel with photocrosslinkable micro-porous networks and a nanoclay component was successfully prepared to control the release of growth factor-rich stem cell secretome. The proven pro-angiogenic and cardioprotective potential of this new bioactive system provides a valuable therapeutic platform for cardiac tissue repair and regeneration. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07806g

  6. Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

    Science.gov (United States)

    Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

    2012-01-01

    It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

  7. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E

    2017-01-01

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effec...

  8. Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus.

    Science.gov (United States)

    Sharma Ghimire, Prakriti; Ouyang, Haomiao; Wang, Qian; Luo, Yuanming; Shi, Bo; Yang, Jinghua; Lü, Yang; Jin, Cheng

    2016-12-02

    Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

  9. Controlled Inhibition of the Mesenchymal Stromal Cell Pro-inflammatory Secretome via Microparticle Engineering

    Directory of Open Access Journals (Sweden)

    Sudhir H. Ranganath

    2016-06-01

    Full Text Available Mesenchymal stromal cells (MSCs are promising therapeutic candidates given their potent immunomodulatory and anti-inflammatory secretome. However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. To address this, we used a microparticle-based engineering approach to non-genetically modulate pro-inflammatory pathways in human MSCs (hMSCs under simulated inflammatory conditions. Here we show that microparticles loaded with TPCA-1, a small-molecule NF-κB inhibitor, when delivered to hMSCs can attenuate secretion of pro-inflammatory factors for at least 6 days in vitro. Conditioned medium (CM derived from TPCA-1-loaded hMSCs also showed reduced ability to attract human monocytes and prevented differentiation of human cardiac fibroblasts to myofibroblasts, compared with CM from untreated or TPCA-1-preconditioned hMSCs. Thus, we provide a broadly applicable bioengineering solution to facilitate intracellular sustained release of agents that modulate signaling. We propose that this approach could be harnessed to improve control over MSC secretome post-transplantation, especially to prevent adverse remodeling post-myocardial infarction.

  10. Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

    OpenAIRE

    Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

    2008-01-01

    The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

  11. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

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    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  12. Molecular Profiling of the Phytophthora plurivora Secretome: A Step towards Understanding the Cross-Talk between Plant Pathogenic Oomycetes and Their Hosts

    Science.gov (United States)

    Fleischmann, Frank; Dalio, Ronaldo J. D.; Di Maro, Antimo; Scognamiglio, Monica; Fiorentino, Antonio; Parente, Augusto; Osswald, Wolfgang; Chambery, Angela

    2014-01-01

    The understanding of molecular mechanisms underlying host–pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora-F. sylvatica interaction

  13. Protein Conformation Ensembles Monitored by HDX Reveal a Structural Rationale for Abscisic Acid Signaling Protein Affinities and Activities

    OpenAIRE

    West, Graham M.; Pascal, Bruce D.; Ng, Ley-Moy; Soon, Fen-Fen; Melcher, Karsten; Xu, H. Eric; Chalmers, Michael J.; Griffin, Patrick R.

    2013-01-01

    Plants regulate growth and respond to environmental stress through abscisic acid (ABA) regulated pathways, and as such these pathways are of primary interest for biological and agricultural research. The ABA response is first perceived by the PYR/PYL/RCAR class of START protein receptors. These ABA activated receptors disrupt phosphatase inhibition of Snf1-related kinases (SnRKs) enabling kinase signaling. Here, insights into the structural mechanism of proteins in the ABA signaling pathway (...

  14. Expressed proteins of Herbaspirillum seropedicae in maize (DKB240) roots-bacteria interaction revealed using proteomics.

    Science.gov (United States)

    Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave

    2014-11-01

    Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.

  15. New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa.

    Directory of Open Access Journals (Sweden)

    Edoardo Giacopuzzi

    Full Text Available Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis, early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals. We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may

  16. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  17. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

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    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  18. Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jingshan [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Nettleship, Joanne E.; Sainsbury, Sarah [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Saunders, Nigel J. [Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Owens, Raymond J., E-mail: ray@strubi.ox.ac.uk [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The X-ray crystal structure of the cold-shock domain protein from N. meningitidis reveals a strand-exchanged dimer. The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 Å resolution and shown to comprise a dimer formed by the exchange of two β-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K{sub d} of 1.25 µM.

  19. Proteomic Analysis Reveals the Deregulation of Inflammation-Related Proteins in Acupuncture-Treated Rats with Asthma Onset

    Directory of Open Access Journals (Sweden)

    Yu-Dong Xu

    2012-01-01

    Full Text Available Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE and mass-spectrometry- (MS- based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11 and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE. These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

  20. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    Science.gov (United States)

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  1. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  2. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  3. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  4. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

    Science.gov (United States)

    Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

    2010-08-03

    Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  5. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins

    DEFF Research Database (Denmark)

    Cappellini, Enrico; Jensen, Lars Juhl; Szklarczyk, Damian Milosz

    2012-01-01

    We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance......We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low......-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African (Loxodonta africana) and Indian (Elephas maximus) elephants. Strong...

  6. Three-Dimensional Bioprinting Nanotechnologies towards Clinical Application of Stem Cells and Their Secretome in Salivary Gland Regeneration

    Directory of Open Access Journals (Sweden)

    Joao N. Ferreira

    2016-01-01

    Full Text Available Salivary gland (SG functional damage and severe dry mouth (or xerostomia are commonly observed in a wide range of medical conditions from autoimmune to metabolic disorders as well as after radiotherapy to treat specific head and neck cancers. No effective therapy has been developed to completely restore the SG functional damage on the long-term and reverse the poor quality of life of xerostomia patients. Cell- and secretome-based strategies are currently being tested in vitro and in vivo for the repair and/or regeneration of the damaged SG using (1 epithelial SG stem/progenitor cells from salispheres or explant cultures as well as (2 nonepithelial stem cell types and/or their bioactive secretome. These strategies will be the focus of our review. Herein, innovative 3D bioprinting nanotechnologies for the generation of organotypic cultures and SG organoids/mini-glands will also be discussed. These bioprinting technologies will allow researchers to analyze the secretome components and extracellular matrix production, as well as their biofunctional effects in 3D mini-glands ex vivo. Improving our understanding of the SG secretome is critical to develop effective secretome-based therapies towards the regeneration and/or repair of all SG compartments for proper restoration of saliva secretion and flow into the oral cavity.

  7. Revealing Linear Aggregates of Light Harvesting Antenna Proteins in Photosynthetic Membranes

    OpenAIRE

    He, Yufan; Zeng, Xiaohua; Mukherjee, Saptarshi; Rajapaksha, Suneth; Kaplan, Samuel; Lu, H. Peter

    2010-01-01

    How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under...

  8. Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

    Science.gov (United States)

    Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

    2016-11-01

    The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

    Science.gov (United States)

    Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

    2013-01-14

    The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Distinct role of hydration water in protein misfolding and aggregation revealed by fluctuating thermodynamics analysis.

    Science.gov (United States)

    Chong, Song-Ho; Ham, Sihyun

    2015-04-21

    Protein aggregation in aqueous cellular environments is linked to diverse human diseases. Protein aggregation proceeds through a multistep process initiated by conformational transitions, called protein misfolding, of monomer species toward aggregation-prone structures. Various forms of aggregate species are generated through the association of misfolded monomers including soluble oligomers and amyloid fibrils. Elucidating the molecular mechanisms and driving forces involved in the misfolding and subsequent association has been a central issue for understanding and preventing protein aggregation diseases such as Alzheimer's, Parkinson's, and type II diabetes. In this Account, we provide a thermodynamic perspective of the misfolding and aggregation of the amyloid-beta (Aβ) protein implicated in Alzheimer's disease through the application of fluctuating thermodynamics. This approach "dissects" the conventional thermodynamic characterization of the end states into the one of the fluctuating processes connecting them, and enables one to analyze variations in the thermodynamic functions that occur during the course of protein conformational changes. The central quantity in this approach is the solvent-averaged effective energy, f = Eu + Gsolv, comprising the protein potential energy (Eu) and the solvation free energy (Gsolv), whose time variation reflects the protein dynamics on the free energy landscape. Protein configurational entropy is quantified by the magnitude of fluctuations in f. We find that misfolding of the Aβ monomer when released from a membrane environment to an aqueous phase is driven by favorable changes in protein potential energy and configurational entropy, but it is also accompanied by an unfavorable increase in solvation free energy. The subsequent dimerization of the misfolded Aβ monomers occurs in two steps. The first step, where two widely separated monomers come into contact distance, is driven by water-mediated attraction, that is, by a

  11. An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS

    DEFF Research Database (Denmark)

    Nielsen, Mari Mæland; Meyer, Sonnich; Larsen, Bodil Katrine

    2011-01-01

    Compound-specific protein expression signatures( PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity,however, there are concerns regarding the reproducibility of this method. The aim of this study was to define...

  12. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    Science.gov (United States)

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Platyhelminth Venom Allergen-Like (VAL) proteins: revealing structural diversity, class-specific features and biological associations across the phylum

    Science.gov (United States)

    CHALMERS, IAIN W.; HOFFMANN, KARL F.

    2012-01-01

    SUMMARY During platyhelminth infection, a cocktail of proteins is released by the parasite to aid invasion, initiate feeding, facilitate adaptation and mediate modulation of the host immune response. Included amongst these proteins is the Venom Allergen-Like (VAL) family, part of the larger sperm coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) superfamily. To explore the significance of this protein family during Platyhelminthes development and host interactions, we systematically summarize all published proteomic, genomic and immunological investigations of the VAL protein family to date. By conducting new genomic and transcriptomic interrogations to identify over 200 VAL proteins (228) from species in all 4 traditional taxonomic classes (Trematoda, Cestoda, Monogenea and Turbellaria), we further expand our knowledge related to platyhelminth VAL diversity across the phylum. Subsequent phylogenetic and tertiary structural analyses reveal several class-specific VAL features, which likely indicate a range of roles mediated by this protein family. Our comprehensive analysis of platyhelminth VALs represents a unifying synopsis for understanding diversity within this protein family and a firm context in which to initiate future functional characterization of these enigmatic members. PMID:22717097

  14. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

    Science.gov (United States)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-08-24

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

  15. Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study

    Energy Technology Data Exchange (ETDEWEB)

    Lesoil, Charles [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Nonaka, Takahiro [Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Sekiguchi, Hiroshi; Osada, Toshiya [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Miyata, Makoto [Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Afrin, Rehana [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Biofrontier Center, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan); Ikai, Atsushi, E-mail: ikai.a.aa@m.titech.ac.jp [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsutacho 4259, Midori-ku, Yokohama 226-8501 (Japan)

    2010-01-15

    Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the 'leg' of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the 'leg' protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

  16. Molecular shape and binding force of Mycoplasma mobile's leg protein Gli349 revealed by an AFM study

    International Nuclear Information System (INIS)

    Lesoil, Charles; Nonaka, Takahiro; Sekiguchi, Hiroshi; Osada, Toshiya; Miyata, Makoto; Afrin, Rehana; Ikai, Atsushi

    2010-01-01

    Recent studies of the gliding bacteria Mycoplasma mobile have identified a family of proteins called the Gli family which was considered to be involved in this novel and yet fairly unknown motility system. The 349 kDa protein called Gli349 was successfully isolated and purified from the bacteria, and electron microscopy imaging and antibody experiments led to the hypothesis that it acts as the 'leg' of M. mobile, responsible for attachment to the substrate as well as for gliding motility. However, more precise evidence of the molecular shape and function of this protein was required to asses this theory any further. In this study, an atomic force microscope (AFM) was used both as an imaging and a force measurement device to provide new information about Gli349 and its role in gliding motility. AFM images of the protein were obtained revealing a complex structure with both rigid and flexible parts, consistent with previous electron micrographs of the protein. Single-molecular force spectroscopy experiments were also performed, revealing that Gli349 is able to specifically bind to sialyllactose molecules and withstand unbinding forces around 70 pN. These findings strongly support the idea that Gli349 is the 'leg' protein of M. mobile, responsible for binding and also most probably force generation during gliding motility.

  17. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    Directory of Open Access Journals (Sweden)

    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  18. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    Directory of Open Access Journals (Sweden)

    Donohue-Rolfe Arthur

    2011-06-01

    Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

  19. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    Science.gov (United States)

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  20. Dynamic changes in protein functional linkage networks revealed by integration with gene expression data.

    Directory of Open Access Journals (Sweden)

    Shubhada R Hegde

    2008-11-01

    Full Text Available Response of cells to changing environmental conditions is governed by the dynamics of intricate biomolecular interactions. It may be reasonable to assume, proteins being the dominant macromolecules that carry out routine cellular functions, that understanding the dynamics of protein:protein interactions might yield useful insights into the cellular responses. The large-scale protein interaction data sets are, however, unable to capture the changes in the profile of protein:protein interactions. In order to understand how these interactions change dynamically, we have constructed conditional protein linkages for Escherichia coli by integrating functional linkages and gene expression information. As a case study, we have chosen to analyze UV exposure in wild-type and SOS deficient E. coli at 20 minutes post irradiation. The conditional networks exhibit similar topological properties. Although the global topological properties of the networks are similar, many subtle local changes are observed, which are suggestive of the cellular response to the perturbations. Some such changes correspond to differences in the path lengths among the nodes of carbohydrate metabolism correlating with its loss in efficiency in the UV treated cells. Similarly, expression of hubs under unique conditions reflects the importance of these genes. Various centrality measures applied to the networks indicate increased importance for replication, repair, and other stress proteins for the cells under UV treatment, as anticipated. We thus propose a novel approach for studying an organism at the systems level by integrating genome-wide functional linkages and the gene expression data.

  1. A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation

    NARCIS (Netherlands)

    Magnani, Enrico; de Klein, Niek; Nam, Hye-In; Kim, Jung-Gun; Pham, Kimberly; Fiume, Elisa; Mudgett, Mary Beth; Rhee, Seung Yon

    2014-01-01

    Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from

  2. Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

    Science.gov (United States)

    Güray, Melda Z; Zheng, Shi; Doucette, Alan A

    2017-02-03

    Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

  3. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

    International Nuclear Information System (INIS)

    Teutschbein, Janka; Haydn, Johannes M; Samans, Birgit; Krause, Michael; Eilers, Martin; Schartl, Manfred; Meierjohann, Svenja

    2010-01-01

    Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

  4. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

    Directory of Open Access Journals (Sweden)

    Krause Michael

    2010-07-01

    Full Text Available Abstract Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1, early growth response 1 (Egr1, osteopontin (Opn, insulin-like growth factor binding protein 3 (Igfbp3, dual-specificity phosphatase 4 (Dusp4, and tumor-associated antigen L6 (Taal6. Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute

  5. Direct observation of TALE protein dynamics reveals a two-state search mechanism.

    Science.gov (United States)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M

    2015-06-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process-a search state and a recognition state-facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

  6. Determination of optimized oxygen partial pressure to maximize the liver regenerative potential of the secretome obtained from adipose-derived stem cells.

    Science.gov (United States)

    Lee, Sang Chul; Kim, Kee-Hwan; Kim, Ok-Hee; Lee, Sang Kuon; Hong, Ha-Eun; Won, Seong Su; Jeon, Sang-Jin; Choi, Byung Jo; Jeong, Wonjun; Kim, Say-June

    2017-08-03

    A hypoxic-preconditioned secretome from stem cells reportedly promotes the functional and regenerative capacity of the liver more effectively than a control secretome. However, the optimum oxygen partial pressure (pO 2 ) in the cell culture system that maximizes the therapeutic potential of the secretome has not yet been determined. We first determined the cellular alterations in adipose tissue-derived stem cells (ASCs) cultured under different pO 2 (21%, 10%, 5%, and 1%). Subsequently, partially hepatectomized mice were injected with the secretome of ASCs cultured under different pO 2 , and then sera and liver specimens were obtained for analyses. Of all AML12 cells cultured under different pO 2 , the AML12 cells cultured under 1% pO 2 showed the highest mRNA expression of proliferation-associated markers (IL-6, HGF, and VEGF). In the cell proliferation assay, the AML12 cells cultured with the secretome of 1% pO 2 showed the highest cell proliferation, followed by the cells cultured with the secretome of 21%, 10%, and 5% pO 2 , in that order. When injected into the partially hepatectomized mice, the 1% pO 2 secretome most significantly increased the number of Ki67-positive cells, reduced serum levels of proinflammatory mediators (IL-6 and TNF-α), and reduced serum levels of liver transaminases. In addition, analysis of the liver specimens indicated that injection with the 1% pO 2 secretome maximized the expression of the intermediate molecules of the PIP3/Akt and IL-6/STAT3 signaling pathways, all of which are known to promote liver regeneration. The data of this study suggest that the secretome of ASCs cultured under 1% pO 2 has the highest liver reparative and regenerative potential of all the secretomes tested here.

  7. Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    2013-01-01

    Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  8. Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

    Science.gov (United States)

    Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

    2016-02-15

    The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications

    DEFF Research Database (Denmark)

    Halim, Adnan; Carlsson, Michael C; Mathiesen, Caroline Benedicte K

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post...... allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic...

  10. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    Science.gov (United States)

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-02

    The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the

  11. Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

    Science.gov (United States)

    Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

    2017-02-15

    Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

  12. Proteomic analysis of Herbaspirillum seropedicae reveals ammonium-induced AmtB-dependent membrane sequestration of PII proteins.

    Science.gov (United States)

    Huergo, Luciano F; Noindorf, Lilian; Gimenes, Camila; Lemgruber, Renato S P; Cordellini, Daniela F; Falarz, Lucas J; Cruz, Leonardo M; Monteiro, Rose A; Pedrosa, Fábio O; Chubatsu, Leda S; Souza, Emanuel M; Steffens, Maria B R

    2010-07-01

    This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

  13. Long-acting beneficial effect of percutaneously intramyocardially delivered secretome of apoptotic peripheral blood cells on porcine chronic ischemic left ventricular dysfunction.

    Science.gov (United States)

    Pavo, Noemi; Zimmermann, Matthias; Pils, Dietmar; Mildner, Michael; Petrási, Zsolt; Petneházy, Örs; Fuzik, János; Jakab, András; Gabriel, Christian; Sipos, Wolfgang; Maurer, Gerald; Gyöngyösi, Mariann; Ankersmit, Hendrik Jan

    2014-04-01

    The quantity of cells with paracrine effects for use in myocardial regeneration therapy is limited. This study investigated the effects of catheter-based endomyocardial delivery of secretome of 2.5 × 10(9) apoptotic peripheral blood mononuclear cells (APOSEC) on porcine chronic post-myocardial infarction (MI) left ventricular (LV) dysfunction and on gene expression. Closed-chest reperfused MI was induced in pigs by 90-min occlusion followed by reperfusion of the mid-LAD (day 0). At day 30, animals were randomized to receive porcine APOSEC (n = 8) or medium solution (control; n = 8) injected intramyocardially into the MI border zone using 3D NOGA guidance. At day 60, cardiac MRI with late enhancement and diagnostic NOGA (myocardial viability) were performed. Gene expression profiling of the infarct core, border zone, and normal myocardium was performed using microarray analysis and confirmed by quantitative real-time PCR. Injection of APOSEC significantly decreased infarct size (p < 0.05) and improved cardiac index and myocardial viability compared to controls. A trend towards higher LV ejection fraction was observed in APOSEC vs. controls (45.4 ± 5.9% vs. 37.4 ± 8.9%, p = 0.052). Transcriptome analysis revealed significant downregulation of caspase-1, tumor necrosis factor and other inflammatory genes in APOSEC-affected areas. rtPCR showed higher expression of myogenic factor Mefc2 (p < 0.05) and downregulated caspase genes (p < 0.05) in APOSEC-treated pigs. In conclusion, overexpression of MEF2c and repression of caspase was related to decreased infarct size and improved cardiac function in secretome-treated animals. Altered gene expression 1-month post-APOSEC treatment proved the long-acting effects of cell-free therapy with paracrine factors. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. Structural fragment clustering reveals novel structural and functional motifs in α-helical transmembrane proteins

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    Vassilev Boris

    2010-04-01

    Full Text Available Abstract Background A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. Results We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to α-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94% appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1 a dimer interface motif found in voltage-gated chloride channels, (2 a proton transfer motif found in heme-copper oxidases, and (3 a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. Conclusions Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes.

  15. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

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    Loren H. Rieseberg

    2012-10-01

    Full Text Available Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq to refine our understanding of the population genomics of wild species of sunflowers (Helianthus spp. and the factors that affect rates of protein evolution. We aligned 35 GB of transcriptome sequencing data and identified 433,257 polymorphic sites (SNPs in a reference transcriptome comprising 16,312 genes. Using SNP markers, we identified strong population clustering largely corresponding to the three species analyzed here (Helianthus annuus, H. petiolaris, H. debilis, with one distinct early generation hybrid. Then, we calculated the proportions of adaptive substitution fixed by selection (alpha and identified gene ontology categories with elevated values of alpha. The “response to biotic stimulus” category had the highest mean alpha across the three interspecific comparisons, implying that natural selection imposed by other organisms plays an important role in driving protein evolution in wild sunflowers. Finally, we examined the relationship between protein evolution (dN/dS ratio and several genomic factors predicted to co-vary with protein evolution (gene expression level, divergence and specificity, genetic divergence [FST], and nucleotide diversity pi. We find that variation in rates of protein divergence was correlated with gene expression level and specificity, consistent with results from a broad range of taxa and timescales. This would in turn imply that these factors govern protein evolution both at a microevolutionary and macroevolutionary timescale. Our results contribute to a general understanding of the

  16. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

    Science.gov (United States)

    Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

    2014-10-01

    Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

  17. Preferential binding effects on protein structure and dynamics revealed by coarse-grained Monte Carlo simulation

    Science.gov (United States)

    Pandey, R. B.; Jacobs, D. J.; Farmer, B. L.

    2017-05-01

    The effect of preferential binding of solute molecules within an aqueous solution on the structure and dynamics of the histone H3.1 protein is examined by a coarse-grained Monte Carlo simulation. The knowledge-based residue-residue and hydropathy-index-based residue-solvent interactions are used as input to analyze a number of local and global physical quantities as a function of the residue-solvent interaction strength (f). Results from simulations that treat the aqueous solution as a homogeneous effective solvent medium are compared to when positional fluctuations of the solute molecules are explicitly considered. While the radius of gyration (Rg) of the protein exhibits a non-monotonic dependence on solvent interaction over a wide range of f within an effective medium, an abrupt collapse in Rg occurs in a narrow range of f when solute molecules rapidly bind to a preferential set of sites on the protein. The structure factor S(q) of the protein with wave vector (q) becomes oscillatory in the collapsed state, which reflects segmental correlations caused by spatial fluctuations in solute-protein binding. Spatial fluctuations in solute binding also modify the effective dimension (D) of the protein in fibrous (D ˜ 1.3), random-coil (D ˜ 1.75), and globular (D ˜ 3) conformational ensembles as the interaction strength increases, which differ from an effective medium with respect to the magnitude of D and the length scale.

  18. Quantum mechanical electronic structure calculation reveals orientation dependence of hydrogen bond energy in proteins.

    Science.gov (United States)

    Mondal, Abhisek; Datta, Saumen

    2017-06-01

    Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge-based potential. Proteins 2017; 85:1046-1055. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Identification of Bombyx mori bidensovirus VD1-ORF4 reveals a novel protein associated with viral structural component.

    Science.gov (United States)

    Li, Guohui; Hu, Zhaoyang; Guo, Xuli; Li, Guangtian; Tang, Qi; Wang, Peng; Chen, Keping; Yao, Qin

    2013-06-01

    Bombyx mori bidensovirus (BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5'-RACE revealed the transcription initiation site of BmBDV ORF4 to be -16 nucleotides upstream from the start codon, and 3'-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion.

  20. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    Science.gov (United States)

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  1. Biological Functions of the Secretome of Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Jan Tommassen

    2017-06-01

    Full Text Available Neisseria meningitidis is a Gram-negative bacterial pathogen that normally resides as a commensal in the human nasopharynx but occasionally causes disease with high mortality and morbidity. To interact with its environment, it transports many proteins across the outer membrane to the bacterial cell surface and into the extracellular medium for which it deploys the common and well-characterized autotransporter, two-partner and type I secretion mechanisms, as well as a recently discovered pathway for the surface exposure of lipoproteins. The surface-exposed and secreted proteins serve roles in host-pathogen interactions, including adhesion to host cells and extracellular matrix proteins, evasion of nutritional immunity imposed by iron-binding proteins of the host, prevention of complement activation, neutralization of antimicrobial peptides, degradation of immunoglobulins, and permeabilization of epithelial layers. Furthermore, they have roles in interbacterial interactions, including the formation and dispersal of biofilms and the suppression of the growth of bacteria competing for the same niche. Here, we will review the protein secretion systems of N. meningitidis and focus on the functions of the secreted proteins.

  2. The salivary secretome of the biting midge, Culicoides sonorensis

    Directory of Open Access Journals (Sweden)

    Christopher J. Lehiy

    2014-06-01

    Full Text Available Culicoides biting midges (Diptera: Ceratopogonidae are hematophagous insects with over 1400 species distributed throughout the world. Many of these species are of particular agricultural importance as primary vectors of bluetongue and Schmallenberg viruses, yet little is known about Culicoides genomics and proteomics. Detailed studies of members from other blood-feeding Dipteran families, including those of mosquito (Culicidae and black fly (Simuliidae, have shown that protein components within the insect’s saliva facilitate the blood feeding process. To determine the protein components in Culicoides sonorensis midges, secreted saliva was collected for peptide sequencing by tandem mass spectrometry. Forty-five secreted proteins were identified, including members of the D7 odorant binding protein family, Kunitz-like serine protease inhibitors, maltase, trypsin, and six novel proteins unique to C. sonorensis. Identifying the complex myriad of proteins in saliva from blood-feeding Dipteran species is critical for understanding their role in blood feeding, arbovirus transmission, and possibly the resulting disease pathogenesis.

  3. Crystal structure of a small heat-shock protein from Xylella fastidiosa reveals a distinct high-order structure.

    Science.gov (United States)

    Fonseca, Emanuella Maria Barreto; Scorsato, Valéria; Dos Santos, Marcelo Leite; Júnior, Atilio Tomazini; Tada, Susely Ferraz Siqueira; Dos Santos, Clelton Aparecido; de Toledo, Marcelo Augusto Szymanski; de Souza, Anete Pereira; Polikarpov, Igor; Aparicio, Ricardo

    2017-04-01

    Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.

  4. The Interactomic Analysis Reveals Pathogenic Protein Networks in Phomopsis longicolla Underlying Seed Decay of Soybean

    Directory of Open Access Journals (Sweden)

    Shuxian Li

    2018-04-01

    Full Text Available Phomopsis longicolla T. W. Hobbs (syn. Diaporthe longicolla is the primary cause of Phomopsis seed decay (PSD in soybean, Glycine max (L. Merrill. This disease results in poor seed quality and is one of the most economically important seed diseases in soybean. The objectives of this study were to infer protein–protein interactions (PPI and to identify conserved global networks and pathogenicity subnetworks in P. longicolla including orthologous pathways for cell signaling and pathogenesis. The interlog method used in the study identified 215,255 unique PPIs among 3,868 proteins. There were 1,414 pathogenicity related genes in P. longicolla identified using the pathogen host interaction (PHI database. Additionally, 149 plant cell wall degrading enzymes (PCWDE were detected. The network captured five different classes of carbohydrate degrading enzymes, including the auxiliary activities, carbohydrate esterases, glycoside hydrolases, glycosyl transferases, and carbohydrate binding molecules. From the PPI analysis, novel interacting partners were determined for each of the PCWDE classes. The most predominant class of PCWDE was a group of 60 glycoside hydrolases proteins. The glycoside hydrolase subnetwork was found to be interacting with 1,442 proteins within the network and was among the largest clusters. The orthologous proteins FUS3, HOG, CYP1, SGE1, and the g5566t.1 gene identified in this study could play an important role in pathogenicity. Therefore, the P. longicolla protein interactome (PiPhom generated in this study can lead to a better understanding of PPIs in soybean pathogens. Furthermore, the PPI may aid in targeting of genes and proteins for further studies of the pathogenicity mechanisms.

  5. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

    Science.gov (United States)

    Sznajder, Anna; Pfeiffer, Daniel; Jendrossek, Dieter

    2015-03-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

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    Mariya Tarazanova

    2017-09-01

    Full Text Available Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities.

  7. Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

    Energy Technology Data Exchange (ETDEWEB)

    Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.; Gritsenko, Marina A.; Jacobs, Jon M.; Smith, Richard D.

    2014-04-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  8. Revealing proteins associated with symbiotic germination of Gastrodia elata by proteomic analysis.

    Science.gov (United States)

    Zeng, Xu; Li, Yuanyuan; Ling, Hong; Chen, Juan; Guo, Shunxing

    2018-03-06

    Gastrodia elata, a mycoheterotrophic orchid, is a well-known medicinal herb. In nature, the seed germination of G. elata requires proper fungal association, because of the absence of endosperm. To germinate successfully, G. elata obtains nutrition from mycorrhizal fungi such as Mycena. However, Mycena is not able to supply nutrition for the further development and enlargement of protocorms into tubers, flowering and fruit setting of G. elata. To date, current genomic studies on this topic are limited. Here we used the proteomic approach to explore changes in G. elata at different stages of symbiotic germination. Using mass spectrometry, 3787 unique proteins were identified, of which 599 were classified as differentially accumulated proteins. Most of these differentially accumulated proteins were putatively involved in energy metabolism, plant defense, molecular signaling, and secondary metabolism. Among them, the defense genes (e.g., pathogenesis-/wound-related proteins, peroxidases, and serine/threonine-protein kinase) were highly expressed in late-stage protocorms, suggesting that fungal colonization triggered the significant defense responses of G. elata. The present study indicated the metabolic change and defensive reaction could disrupt the balance between Mycena and G. elata during mycorrhizal symbiotic germination.

  9. Cell Surface Properties of Lactococcus lactis Reveal Milk Protein Binding Specifically Evolved in Dairy Isolates

    Science.gov (United States)

    Tarazanova, Mariya; Huppertz, Thom; Beerthuyzen, Marke; van Schalkwijk, Saskia; Janssen, Patrick; Wels, Michiel; Kok, Jan; Bachmann, Herwig

    2017-01-01

    Surface properties of bacteria are determined by the molecular composition of the cell wall and they are important for interactions of cells with their environment. Well-known examples of bacterial interactions with surfaces are biofilm formation and the fermentation of solid materials like food and feed. Lactococcus lactis is broadly used for the fermentation of cheese and buttermilk and it is primarily isolated from either plant material or the dairy environment. In this study, we characterized surface hydrophobicity, charge, emulsification properties, and the attachment to milk proteins of 55 L. lactis strains in stationary and exponential growth phases. The attachment to milk protein was assessed through a newly developed flow cytometry-based protocol. Besides finding a high degree of biodiversity, phenotype-genotype matching allowed the identification of candidate genes involved in the modification of the cell surface. Overexpression and gene deletion analysis allowed to verify the predictions for three identified proteins that altered surface hydrophobicity and attachment of milk proteins. The data also showed that lactococci isolated from a dairy environment bind higher amounts of milk proteins when compared to plant isolates. It remains to be determined whether the alteration of surface properties also has potential to alter starter culture functionalities. PMID:28936202

  10. Identification of Proteins Using iTRAQ and Virus-Induced Gene Silencing Reveals Three Bread Wheat Proteins Involved in the Response to Combined Osmotic-Cold Stress.

    Science.gov (United States)

    Zhang, Ning; Zhang, Lingran; Shi, Chaonan; Zhao, Lei; Cui, Dangqun; Chen, Feng

    2018-05-25

    Crops are often subjected to a combination of stresses in the field. To date, studies on the physiological and molecular responses of common wheat to a combination of osmotic and cold stresses, however, remain unknown. In this study, wheat seedlings exposed to osmotic-cold stress for 24 h showed inhibited growth, as well as increased lipid peroxidation, relative electrolyte leakage, and soluble sugar contents. iTRAQ-based quantitative proteome method was employed to determine the proteomic profiles of the roots and leaves of wheat seedlings exposed to osmotic-cold stress conditions. A total of 250 and 258 proteins with significantly altered abundance in the roots and leaves were identified, respectively, and the majority of these proteins displayed differential abundance, thereby revealing organ-specific differences in adaptation to osmotic-cold stress. Yeast two hybrid assay examined five pairs of stress/defense-related protein-protein interactions in the predicted protein interaction network. Furthermore, quantitative real-time PCR analysis indicated that abiotic stresses increased the expression of three candidate protein genes, i.e., TaGRP2, CDCP, and Wcor410c in wheat leaves. Virus-induced gene silencing indicated that three genes TaGRP2, CDCP, and Wcor410c were involved in modulating osmotic-cold stress in common wheat. Our study provides useful information for the elucidation of molecular and genetics bases of osmotic-cold combined stress in bread wheat.

  11. Reaction trajectory revealed by a joint analysis of protein data bank.

    Science.gov (United States)

    Ren, Zhong

    2013-01-01

    Structural motions along a reaction pathway hold the secret about how a biological macromolecule functions. If each static structure were considered as a snapshot of the protein molecule in action, a large collection of structures would constitute a multidimensional conformational space of an enormous size. Here I present a joint analysis of hundreds of known structures of human hemoglobin in the Protein Data Bank. By applying singular value decomposition to distance matrices of these structures, I demonstrate that this large collection of structural snapshots, derived under a wide range of experimental conditions, arrange orderly along a reaction pathway. The structural motions along this extensive trajectory, including several helical transformations, arrive at a reverse engineered mechanism of the cooperative machinery (Ren, companion article), and shed light on pathological properties of the abnormal homotetrameric hemoglobins from α-thalassemia. This method of meta-analysis provides a general approach to structural dynamics based on static protein structures in this post genomics era.

  12. Reaction trajectory revealed by a joint analysis of protein data bank.

    Directory of Open Access Journals (Sweden)

    Zhong Ren

    Full Text Available Structural motions along a reaction pathway hold the secret about how a biological macromolecule functions. If each static structure were considered as a snapshot of the protein molecule in action, a large collection of structures would constitute a multidimensional conformational space of an enormous size. Here I present a joint analysis of hundreds of known structures of human hemoglobin in the Protein Data Bank. By applying singular value decomposition to distance matrices of these structures, I demonstrate that this large collection of structural snapshots, derived under a wide range of experimental conditions, arrange orderly along a reaction pathway. The structural motions along this extensive trajectory, including several helical transformations, arrive at a reverse engineered mechanism of the cooperative machinery (Ren, companion article, and shed light on pathological properties of the abnormal homotetrameric hemoglobins from α-thalassemia. This method of meta-analysis provides a general approach to structural dynamics based on static protein structures in this post genomics era.

  13. Autonomously folding protein fragments reveal differences in the energy landscapes of homologous RNases H.

    Directory of Open Access Journals (Sweden)

    Laura E Rosen

    Full Text Available An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare E. coli and T. thermophilus homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I(core and I(core+1 hypothesized to be present on the energy landscapes of these two proteins. We observe that both T. thermophilus RNase H (ttRNH fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two E. coli RNase H (ecRNH fragments have very similar stabilities, suggesting that the presence of additional residues in the I(core+1 fragment does not affect the folding or structure as compared to I(core. NMR experiments provide additional evidence that only the I(core intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I(core+1 intermediate. We determine that the ttRNH I(core+1 intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to E. coli RNase H for which I(core is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.

  14. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

    Directory of Open Access Journals (Sweden)

    D.A. Meireles

    2017-08-01

    Full Text Available Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species. Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr, the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols, but not by β-mercaptoethanol or GSH (monothiols, even in large excess; (ii MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH over hydrogen peroxide; (iii MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13×108 M−1 s−1. Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Keywords: Ohr/OsmC, Thiol-dependent peroxidases, Phylogeny

  15. Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi.

    Science.gov (United States)

    Meireles, D A; Domingos, R M; Gaiarsa, J W; Ragnoni, E G; Bannitz-Fernandes, R; da Silva Neto, J F; de Souza, R F; Netto, L E S

    2017-08-01

    Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×10 8 M -1 s -1 ). Both Cys 87 and Cys 154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pK a value of the Cys p residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Copyright © 2017. Published by Elsevier B.V.

  16. Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

    Science.gov (United States)

    Yoshida, Masa-aki; Yamada, Lixy; Ochi, Hiroe; Iwata, Yoko; Tamura-Nakano, Miwa; Sawada, Hitoshi; Sauer, Warwick H H; Ogura, Atsushi; Hirohashi, Noritaka

    2014-08-01

    In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  18. In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

    Science.gov (United States)

    Fang, Pan; Wang, Xin-Jian; Xue, Yu; Liu, Ming-Qi; Zeng, Wen-Feng; Zhang, Yang; Zhang, Lei; Gao, Xing; Yan, Guo-Quan; Yao, Jun; Shen, Hua-Li; Yang, Peng-Yuan

    2016-06-21

    N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

  19. Exoproteome Analysis of the Seaweed Pathogen Nautella italica R11 Reveals Temperature-Dependent Regulation of RTX-Like Proteins

    Directory of Open Access Journals (Sweden)

    Melissa Gardiner

    2017-06-01

    Full Text Available Climate fluctuations have been linked to an increased prevalence of disease in seaweeds, including the red alga Delisea pulchra, which is susceptible to a bleaching disease caused by the bacterium Nautella italica R11 under elevated seawater temperatures. To further investigate the role of temperature in the induction of disease by N. italica R11, we assessed the effect of temperature on the expression of the extracellular proteome (exoproteome in this bacterium. Label-free quantitative mass spectrometry was used to identify 207 proteins secreted into supernatant fraction, which is equivalent to 5% of the protein coding genes in the N. italica R11 genome. Comparative analysis demonstrated that expression of over 30% of the N. italica R11 exoproteome is affected by temperature. The temperature-dependent proteins include traits that could facilitate the ATP-dependent transport of amino acid and carbohydrate, as well as several uncharacterized proteins. Further, potential virulence determinants, including two RTX-like proteins, exhibited significantly higher expression in the exoproteome at the disease inducing temperature of 24°C relative to non-inducing temperature (16°C. This is the first study to demonstrate that temperature has an influence exoproteome expression in a macroalgal pathogen. The results have revealed several temperature regulated candidate virulence factors that may have a role in macroalgal colonization and invasion at elevated sea-surface temperatures, including novel RTX-like proteins.

  20. Omic studies reveal the pathogenic lipid droplet proteins in non-alcoholic fatty liver disease

    Directory of Open Access Journals (Sweden)

    Xuelin Zhang

    2016-10-01

    Full Text Available Abstract Non-alcoholic fatty liver disease (NAFLD is an epidemic metabolic condition driven by an underlying lipid homeostasis disorder. The lipid droplet (LD, the main organelle involved in neutral lipid storage and hydrolysis, is a potential target for NAFLD therapeutic treatment. In this review, we summarize recent progress elucidating the connections between LD-associated proteins and NAFLD found by genome-wide association studies (GWAS, genomic and proteomic studies. Finally, we discuss a possible mechanism by which the protein 17β-hydroxysteroid dehydrogenase 13 (17β-HSD13 may promote the development of NAFLD.

  1. Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

    Science.gov (United States)

    Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

    1988-12-01

    Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

  2. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    Science.gov (United States)

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  3. SILAC-based comparative analysis of pathogenic Escherichia coli secretomes

    DEFF Research Database (Denmark)

    Boysen, Anders; Borch, Jonas; Krogh, Thøger Jensen

    2015-01-01

    Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence...... experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the colonization factor YghJ and the surface adhesin antigen 43, which is involved in pathogenesis of other Gram-negative bacteria......-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative...

  4. Comparative studies of the secretome of fungus-growing ants

    DEFF Research Database (Denmark)

    Linde, Tore; Grell, Morten Nedergaard; Schiøtt, Morten

    2009-01-01

    Leafcutter ants of the species Acromyrmex echinatior live in symbiosis with the fungus Leucoagaricus gongylophorus. The ants harvest fragments of leaves and carry them to the nest where they place the material on the fungal colony. The fungus secretes a wide array of proteins to degrade the leaves...... into nutrients that the ants can feed on. The focus of this study is to discover, characterize and compare the secreted proteins. In order to do so cDNA libraries are constructed from mRNA extracted from the fungus material. The most efficient technology to screen cDNA libraries selectively for secreted...

  5. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  6. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  7. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    International Nuclear Information System (INIS)

    Khotin, Mikhail; Turoverova, Lidia; Aksenova, Vasilisa; Barlev, Nikolai; Borutinskaite, Veronika Viktorija; Vener, Alexander; Bajenova, Olga; Magnusson, Karl-Eric; Pinaev, George P.; Tentler, Dmitri

    2010-01-01

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  8. Concordance of gene expression in human protein complexes reveals tissue specificity and pathology

    DEFF Research Database (Denmark)

    Börnigen, Daniela; Pers, Tune Hannes; Thorrez, Lieven

    2013-01-01

    Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among prot...

  9. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

    DEFF Research Database (Denmark)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen

    2008-01-01

    , there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14)C content from the atmospheric carbon dioxide. The (14)C content of the lens proteins thus reflects the atmospheric content of (14)C when the lens crystallines were formed. Precise radiocarbon...

  10. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  11. Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy

    NARCIS (Netherlands)

    Groot, M.L.; van Wilderen, L.; Larsen, D.S.; Horst, M.A.; van Stokkum, I.H.M.; Hellingwerf, K.J.; van Grondelle, R.

    2003-01-01

    Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here

  12. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

  13. Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

    Science.gov (United States)

    Hilton, Nicholas A; Sladewski, Thomas E; Perry, Jenna A; Pataki, Zemplen; Sinclair-Davis, Amy N; Muniz, Richard S; Tran, Holly L; Wurster, Jenna I; Seo, Jiwon; de Graffenried, Christopher L

    2018-05-21

    The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly-polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely-configured process in kinetoplastids. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

  14. Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Haijun [Washington University; Zhang, Hao [Washington University; King, Jeremy D. [Washington University; Wolf, Nathan R. [Washington University; Prado, Mindy [Washington University; Gross, Michael L. [Washington University; Blankenship, Robert E. [Washington University

    2014-12-01

    The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

  15. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

    2013-01-22

    Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  16. Comment on "Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry".

    Science.gov (United States)

    Pevzner, Pavel A; Kim, Sangtae; Ng, Julio

    2008-08-22

    Asara et al. (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.

  17. Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

    NARCIS (Netherlands)

    Lai, Xuelei; Wichers, Harry J.; Soler-Lopez, Montserrat; Dijkstra, Bauke W.

    2017-01-01

    Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the

  18. Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells.

    Science.gov (United States)

    Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun

    2014-07-23

    The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Functional genomics of tick thioester-containing proteins reveal the ancient origin of the complement system

    Czech Academy of Sciences Publication Activity Database

    Burešová, Veronika; Hajdušek, Ondřej; Franta, Zdeněk; Loosová, Gabriela; Grunclová, Lenka; Levashina, E.A.; Kopáček, Petr

    2011-01-01

    Roč. 3, č. 6 (2011), s. 623-630 ISSN 1662-811X R&D Projects: GA ČR GAP506/10/2136; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518 Keywords : tick * thioester-containing proteins * complement Subject RIV: EC - Immunology Impact factor: 4.209, year: 2011

  20. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Czech Academy of Sciences Publication Activity Database

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    2016-01-01

    Roč. 132, č. 1 (2016), s. 13-20 ISSN 1874-3919 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.914, year: 2016

  1. Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

    Science.gov (United States)

    Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

    2014-11-01

    Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  2. Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development.

    Science.gov (United States)

    Obudulu, Ogonna; Bygdell, Joakim; Sundberg, Björn; Moritz, Thomas; Hvidsten, Torgeir R; Trygg, Johan; Wingsle, Gunnar

    2016-02-18

    Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease

  3. The crystal structure of the Dachshund domain of human SnoN reveals flexibility in the putative protein interaction surface.

    Directory of Open Access Journals (Sweden)

    Tomas Nyman

    2010-09-01

    Full Text Available The human SnoN is an oncoprotein that interacts with several transcription-regulatory proteins such as the histone-deacetylase, N-CoR containing co-repressor complex and Smad proteins. This study presents the crystal structure of the Dachshund homology domain of human SnoN. The structure reveals a groove composed of conserved residues with characteristic properties of a protein-interaction surface. A comparison of the 12 monomers in the asymmetric unit reveals the presence of two major conformations: an open conformation with a well accessible groove and a tight conformation with a less accessible groove. The variability in the backbone between the open and the tight conformations matches the differences seen in previously determined structures of individual Dachshund homology domains, suggesting a general plasticity within this fold family. The flexibility observed in the putative protein binding groove may enable SnoN to recognize multiple interaction partners.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  4. Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sylvie Cornelie

    Full Text Available Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

  5. Structural organization of the genes for rat von Ebner's gland proteins 1 and 2 reveals their close relationship to lipocalins.

    Science.gov (United States)

    Kock, K; Ahlers, C; Schmale, H

    1994-05-01

    The rat von Ebner's gland protein 1 (VEGP 1) is a secretory protein, which is abundantly expressed in the small acinar von Ebner's salivary glands of the tongue. Based on the primary structure of this protein we have previously suggested that it is a member of the lipocalin superfamily of lipophilic-ligand carrier proteins. Although the physiological role of VEGP 1 is not clear, it might be involved in sensory or protective functions in the taste epithelium. Here, we report the purification of VEGP 1 and of a closely related secretory polypeptide, VEGP 2, the isolation of a cDNA clone encoding VEGP 2, and the isolation and structural characterization of the genes for both proteins. Protein purification by gel-filtration and anion-exchange chromatography using Mono Q revealed the presence of two different immunoreactive VEGP species. N-terminal sequence determination of peptide fragments isolated after protease Asp-N digestion allowed the identification of a new VEGP, named VEGP 2, in addition to the previously characterized VEGP 1. The complete VEGP 2 sequence was deduced from a cDNA clone isolated from a von Ebner's gland cDNA library. The VEGP 2 cDNA encodes a protein of 177 amino acids and is 94% identical to VEGP 1. DNA sequence analysis of the rat VEGP 1 and 2 genes isolated from rat genomic libraries revealed that both span about 4.5 kb and contain seven exons. The VEGP 1 and 2 genes are non-allelic distinct genes in the rat genome and probably arose by gene duplication. The high degree of nucleotide sequence identity in introns A-C (94-100%) points to a recent gene conversion event that included the 5' part of the genes. The genomic organization of the rat VEGP genes closely resembles that found in other lipocalins such as beta-lactoglobulin, mouse urinary proteins (MUPs) and prostaglandin D synthase, and therefore provides clear evidence that VEGPs belong to this superfamily of proteins.

  6. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  7. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    Energy Technology Data Exchange (ETDEWEB)

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  8. Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

    Science.gov (United States)

    Rodero, Mathieu P; Decalf, Jérémie; Bondet, Vincent; Hunt, David; Rice, Gillian I; Werneke, Scott; McGlasson, Sarah L; Alyanakian, Marie-Alexandra; Bader-Meunier, Brigitte; Barnerias, Christine; Bellon, Nathalia; Belot, Alexandre; Bodemer, Christine; Briggs, Tracy A; Desguerre, Isabelle; Frémond, Marie-Louise; Hully, Marie; van den Maagdenberg, Arn M J M; Melki, Isabelle; Meyts, Isabelle; Musset, Lucile; Pelzer, Nadine; Quartier, Pierre; Terwindt, Gisela M; Wardlaw, Joanna; Wiseman, Stewart; Rieux-Laucat, Frédéric; Rose, Yoann; Neven, Bénédicte; Hertel, Christina; Hayday, Adrian; Albert, Matthew L; Rozenberg, Flore; Crow, Yanick J; Duffy, Darragh

    2017-05-01

    Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation. © 2017 Rodero et al.

  9. In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

    Directory of Open Access Journals (Sweden)

    Jeremiah Athmer

    2017-01-01

    Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

  10. Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

    Science.gov (United States)

    Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

    2017-08-08

    Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

  11. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding.

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  12. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  13. Interactions among the early Escherichia coli divisome proteins revealed by bimolecular fluorescence complementation.

    Science.gov (United States)

    Pazos, Manuel; Natale, Paolo; Margolin, William; Vicente, Miguel

    2013-12-01

    We used bimolecular fluorescence complementation (BiFC) assays to detect protein-protein interactions of all possible pairs of the essential Escherichia coli proto-ring components, FtsZ, FtsA and ZipA, as well as the non-essential FtsZ-associated proteins ZapA and ZapB. We found an unexpected interaction between ZipA and ZapB at potential cell division sites, and when co-overproduced, they induced long narrow constrictions at division sites that were dependent on FtsZ. These assays also uncovered an interaction between ZipA and ZapA that was mediated by FtsZ. BiFC with ZapA and ZapB showed that in addition to their expected interaction at midcell, they also interact at the cell poles. BiFC detected interaction between FtsZ and ZapB at midcell and close to the poles. Results from the remaining pairwise combinations confirmed known interactions between FtsZ and ZipA, and ZapB with itself. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  14. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization.

    Directory of Open Access Journals (Sweden)

    Valeriy Demchev

    Full Text Available Fibrinogen like protein 1(Fgl1 is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration.

  15. Toscana virus induces interferon although its NSs protein reveals antagonistic activity.

    Science.gov (United States)

    Gori Savellini, Gianni; Weber, Friedemann; Terrosi, Chiara; Habjan, Matthias; Martorelli, Barbara; Cusi, Maria Grazia

    2011-01-01

    Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-β), although its non-structural protein (NSs) could inhibit the induction of IFN-β if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-β expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-β transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

  16. Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Julie Bachmann

    2014-04-01

    Full Text Available Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.

  17. Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

    Science.gov (United States)

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-02-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

  18. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    International Nuclear Information System (INIS)

    Mulari, Mika T.K.; Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-01-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-β-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption

  19. Organ tropic metastatic secretomes and exosomes in breast cancer

    Science.gov (United States)

    2017-10-01

    biomolecules (i.e., proteins, lipids , RNAs, DNA) contained by exosomes can be horizontally transferred to recipient cells. We showed that an “exosomal...nanoparticles not previously described, which were smaller than 50 nm (~35nm) and clearly lacked an external membrane structure (Fig. 1c); we therefore named...these structures “exomeres”. The other two nanoparticle subpopulations we refer to as small exosomes (Exo-S; 60-80nm [P3]) and large exosomes (Exo-L

  20. Secretome within the bone marrow microenvironment: A basis for mesenchymal stem cell treatment and role in cancer dormancy.

    Science.gov (United States)

    Eltoukhy, Hussam S; Sinha, Garima; Moore, Caitlyn; Gergues, Marina; Rameshwar, Pranela

    2018-05-31

    The secretome produced by cells within the bone marrow is significant to homeostasis. The bone marrow, a well-studied organ, has multiple niches with distinct roles for supporting stem cell functions. Thus, an understanding of mediators involved in the regulation of stem cells could serve as a model for clinical problems and solutions such as tissue repair and regeneration. The exosome secretome of bone marrow stem cells is a developing area of research with respect to the regenerative potential by bone marrow cell, particularly the mesenchymal stem cells. The bone marrow niche regulates endogenous processes such as hematopoiesis but could also support the survival of tumors such as facilitating the cancer stem cells to exist in dormancy for decades. The bone marrow-derived secretome will be critical to future development of therapeutic strategies for oncologic diseases, in addition to regenerative medicine. This article discusses the importance for parallel studies to determine how the same secretome may compromise safety during the use of stem cells in regenerative medicine. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Genome, transcriptome, and secretome analysis of wood decay fungus postia placenta supports unique mechanisms of lignocellulose conversion

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Diego [Los Alamos National Laboratory; Challacombe, Jean F [Los Alamos National Laboratory; Misra, Monica [Los Alamos National Laboratory; Xie, Gary [Los Alamos National Laboratory; Brettin, Thomas [Los Alamos National Laboratory; Morgenstern, Ingo [CLARK UNIV; Hibbett, David [CLARK UNIV.; Schmoll, Monika [UNIV WIEN; Kubicek, Christian P [UNIV WIEN; Ferreira, Patricia [CIB, CSIC, MADRID; Ruiz - Duenase, Francisco J [CIB, CSIC, MADRID; Martinez, Angel T [CIB, CSIC, MADRID; Kersten, Phil [FOREST PRODUCTS LAB; Hammel, Kenneth E [FOREST PRODUCTS LAB; Vanden Wymelenberg, Amber [U. WISCONSIN; Gaskell, Jill [FOREST PRODUCTS LAB; Lindquist, Erika [DOE JGI; Sabati, Grzegorz [U. WISCONSIN; Bondurant, Sandra S [U. WISCONSIN; Larrondo, Luis F [U. CATHOLICA DE CHILE; Canessa, Paulo [U. CATHOLICA DE CHILE; Vicunna, Rafael [U. CATHOLICA DE CHILE; Yadavk, Jagiit [U. CINCINATTI; Doddapaneni, Harshavardhan [U. CINCINATTI; Subramaniank, Venkataramanan [U. CINCINATTI; Pisabarro, Antonio G [PUBLIC U. NAVARRE; Lavin, Jose L [PUBLIC U. NAVARRE; Oguiza, Jose A [PUBLIC U. NAVARRE; Master, Emma [U. TORONTO; Henrissat, Bernard [CNRS, MARSEILLE; Coutinho, Pedro M [CNRS, MARSEILLE; Harris, Paul [NOVOZYMES, INC.; Magnuson, Jon K [PNNL; Baker, Scott [PNNL; Bruno, Kenneth [PNNL; Kenealy, William [MASCOMA, INC.; Hoegger, Patrik J [GEORG-AUGUST-U.; Kues, Ursula [GEORG-AUGUST-U; Ramaiva, Preethi [NOVOZYMES, INC.; Lucas, Susan [DOE JGI; Salamov, Asaf [DOE JGI; Shapiro, Harris [DOE JGI; Tuh, Hank [DOE JGI; Chee, Christine L [UNM; Teter, Sarah [NOVOZYMES, INC.; Yaver, Debbie [NOVOZYMES, INC.; James, Tim [MCMASTER U.; Mokrejs, Martin [CHARLES U.; Pospisek, Martin [CHARLES U.; Grigoriev, Igor [DOE JGI; Rokhsar, Dan [DOE JGI; Berka, Randy [NOVOZYMES; Cullen, Dan [FOREST PRODUCTS LAB

    2008-01-01

    Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative {beta}-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC{center_dot}MSIMS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H202. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H202 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons to the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

  2. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  3. A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

    Science.gov (United States)

    Schaechinger, Thorsten J; Gorbunov, Dmitry; Halaszovich, Christian R; Moser, Tobias; Kügler, Sebastian; Fakler, Bernd; Oliver, Dominik

    2011-06-24

    Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

  4. Protein dynamics revealed in the excitonic spectra of single LH2 complexes

    International Nuclear Information System (INIS)

    Valkunas, Leonas; Janusonis, Julius; Rutkauskas, Danielis; Grondelle, Rienk van

    2007-01-01

    The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

  5. Survey of large protein complexes D. vulgaris reveals great structural diversity

    Energy Technology Data Exchange (ETDEWEB)

    Han, B.-G.; Dong, M.; Liu, H.; Camp, L.; Geller, J.; Singer, M.; Hazen, T. C.; Choi, M.; Witkowska, H. E.; Ball, D. A.; Typke, D.; Downing, K. H.; Shatsky, M.; Brenner, S. E.; Chandonia, J.-M.; Biggin, M. D.; Glaeser, R. M.

    2009-08-15

    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr {approx} 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate {approx}10 times greater than that of previous 'proteomic' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

  6. Internal jugular vein thrombosis associated with venous hypoplasia and protein S deficiency revealed by ultrasonography.

    Science.gov (United States)

    Lim, Byung Gun; Kim, Young Min; Kim, Heezoo; Lim, Sang Ho; Lee, Mi Kyoung

    2011-12-01

    A 41-year-old woman, who had no thrombotic risk factors and past history except congenital scoliosis, underwent central venous catheterization (CVC) before correction of the scoliosis. When internal jugular vein (IJV) catheterization using the anatomical landmark technique failed, CVC under ultrasound guidance was tried. As a consequence, thrombosis and hypoplasia of the right IJV were incidentally detected by ultrasonography. Central venous catheters were then successfully placed in other veins under ultrasound guidance. Also, after examinations to rule out the possibility of pulmonary embolism and to clarify the causes of the IJV thrombosis, the patient was found to have protein S deficiency. CVC under ultrasound guidance should be recommended to prevent the failure of cannulation and complications such as thromboembolism in patients who could possibly have anomalies of vessels as a result of anatomical deformities caused by severe scoliosis, even if patients do not have thrombotic risk factors such as a history of central catheter insertion or intravenous drug abuse, cancer, advanced age, cerebral infarction, and left ventricular dysfunction. Also, if venous thrombosis is found in patients without predisposing risk factors, one should ascertain the cause of the hypercoagulable state, for example protein S deficiency, and perform appropriate treatment and prevention of venous thromboembolism.

  7. Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

    Science.gov (United States)

    Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

    2016-03-01

    Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

  8. Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36

    Directory of Open Access Journals (Sweden)

    Stephanie M. Bywaters

    2017-11-01

    Full Text Available The currently available nonavalent human papillomavirus (HPV vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs (H16.V5, H16.U4 and H16.7E and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17–36. The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

  9. Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17-36.

    Science.gov (United States)

    Bywaters, Stephanie M; Brendle, Sarah A; Tossi, Kerstin P; Biryukov, Jennifer; Meyers, Craig; Christensen, Neil D

    2017-11-10

    The currently available nonavalent human papillomavirus (HPV) vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP) vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs) (H16.V5, H16.U4 and H16.7E) and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17-36). The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

  10. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

    Science.gov (United States)

    Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

    2013-11-19

    JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

  11. Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

    Science.gov (United States)

    Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

    2018-04-10

    Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

  12. A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

    Science.gov (United States)

    DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

    2014-07-15

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. © 2014 DeMille et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

    2005-01-01

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  14. Proteomic analysis of tissue from α1,3-galactosyltransferase knockout mice reveals that a wide variety of proteins and protein fragments change expression level.

    Directory of Open Access Journals (Sweden)

    Louise Thorlacius-Ussing

    Full Text Available A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient's blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes.

  15. Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

    Science.gov (United States)

    Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

    2015-05-01

    Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

  16. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  17. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Lisby, Michael; Altmannova, Veronika

    2009-01-01

    , and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2......Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 "anti-recombinase" restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we...... removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR....

  18. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  19. Structures of Adnectin/Protein Complexes Reveal an Expanded Binding Footprint

    Energy Technology Data Exchange (ETDEWEB)

    Ramamurthy, Vidhyashankar; Krystek, Jr., Stanley R.; Bush, Alexander; Wei, Anzhi; Emanuel, Stuart L.; Gupta, Ruchira Das; Janjua, Ahsen; Cheng, Lin; Murdock, Melissa; Abramczyk, Bozena; Cohen, Daniel; Lin, Zheng; Morin, Paul; Davis, Jonathan H.; Dabritz, Michael; McLaughlin, Douglas C.; Russo, Katie A.; Chao, Ginger; Wright, Martin C.; Jenny, Victoria A.; Engle, Linda J.; Furfine, Eric; Sheriff, Steven (BMS)

    2014-10-02

    Adnectins are targeted biologics derived from the tenth type III domain of human fibronectin ({sup 10}Fn3), a member of the immunoglobulin superfamily. Target-specific binders are selected from libraries generated by diversifying the three {sup 10}Fn3 loops that are analogous to the complementarity determining regions of antibodies. The crystal structures of two Adnectins were determined, each in complex with its therapeutic target, EGFR or IL-23. Both Adnectins bind different epitopes than those bound by known monoclonal antibodies. Molecular modeling suggests that some of these epitopes might not be accessible to antibodies because of the size and concave shape of the antibody combining site. In addition to interactions from the Adnectin diversified loops, residues from the N terminus and/or the {beta} strands interact with the target proteins in both complexes. Alanine-scanning mutagenesis confirmed the calculated binding energies of these {beta} strand interactions, indicating that these nonloop residues can expand the available binding footprint.

  20. Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

    Directory of Open Access Journals (Sweden)

    Ardizzone Michele

    2008-08-01

    Full Text Available Abstract Background The present study has investigated the protein tyrosine phosphatase H1 (PTPH1 expression pattern in mouse brain and its impact on CNS functions. Methods We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype were also behaviorally tested for CNS functions. Results In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

  1. The Structure of the Poxvirus A33 Protein Reveals a Dimer of Unique C-Type Lectin-Like Domains

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hua-Poo; Singh, Kavita; Gittis, Apostolos G.; Garboczi, David N. (NIH)

    2010-11-03

    The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

  2. Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

    Science.gov (United States)

    Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

    2011-04-01

    The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

  3. Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

    Science.gov (United States)

    McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

    2016-01-01

    Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

  4. A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth.

    Science.gov (United States)

    Viloria, Katrina; Munasinghe, Amanda; Asher, Sharan; Bogyere, Roberto; Jones, Lucy; Hill, Natasha J

    2016-11-25

    SPARC is a matricellular protein that is involved in both pancreatic cancer and diabetes. It belongs to a wider family of proteins that share structural and functional similarities. Relatively little is known about this extended family, but evidence of regulatory interactions suggests the importance of a holistic approach to their study. We show that Hevin, SPOCKs, and SMOCs are strongly expressed within islets, ducts, and blood vessels, suggesting important roles for these proteins in the normal pancreas, while FSTL-1 expression is localised to the stromal compartment reminiscent of SPARC. In direct contrast to SPARC, however, FSTL-1 expression is reduced in pancreatic cancer. Consistent with this, FSTL-1 inhibited pancreatic cancer cell proliferation. The complexity of SPARC family proteins is further revealed by the detection of multiple cell-type specific isoforms that arise due to a combination of post-translational modification and alternative splicing. Identification of splice variants lacking a signal peptide suggests the existence of novel intracellular isoforms. This study underlines the importance of addressing the complexity of the SPARC family and provides a new framework to explain their controversial and contradictory effects. We also demonstrate for the first time that FSTL-1 suppresses pancreatic cancer cell growth.

  5. Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

    Science.gov (United States)

    Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

    2018-06-03

    Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

  6. Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

    Science.gov (United States)

    Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

    2017-04-28

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

  7. Fibrillation mechanism of a model intrinsically disordered protein revealed by 2D correlation deep UV resonance Raman spectroscopy.

    Science.gov (United States)

    Sikirzhytski, Vitali; Topilina, Natalya I; Takor, Gaius A; Higashiya, Seiichiro; Welch, John T; Uversky, Vladimir N; Lednev, Igor K

    2012-05-14

    Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of β-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and β-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.

  8. Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

    Directory of Open Access Journals (Sweden)

    Ogata Hiroyuki

    2011-09-01

    Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

  9. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen

    2011-01-01

    . In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins...... in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates....... Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes....

  10. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  11. Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

    Science.gov (United States)

    Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

    2015-10-14

    than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

  12. Systemic administration of 3-bromopyruvate reveals its interaction with serum proteins in a rat model.

    Science.gov (United States)

    Kunjithapatham, Rani; Geschwind, Jean-Francois H; Rao, Pramod P; Boronina, Tatiana N; Cole, Robert N; Ganapathy-Kanniappan, Shanmugasundaram

    2013-07-17

    3-bromopyruvate (3-BrPA) is a glycolytic inhibitor that affects cancer cells by targeting energy metabolism. Preclinical reports have established that a 1.75 mM dose of 3-BrPA is effective and sufficient to inhibit tumor growth when administered under a loco-regional approach (intraarterial and intratumoral). This loco-regional therapeutic dose was found to be nontoxic when given systemically as well. Yet, the mechanism underlying this lack of toxicity of 1.75 mM 3-BrPA during systemic delivery is unknown. Here, we investigated the mechanism associated with the lack of organ toxicity when 1.75 mM 3-BrPA was administered systemically using radiolabeled (14C)-3-BrPA in Sprague-Dawley rats. Data obtained from tissue-autoradiography of rats infused with 14C-3-BrPA showed strong 14C-signal in tissue sections of various organs except the brain corroborating that 3-BrPA does not cross the blood-brain barrier. Significantly, Hematoxylin & Eosin staining and apoptosis assay of tissue sections positive for 14C-signal showed no signs of toxicity or apoptosis. Convincingly, the 14C-signal observed in tissue-autoradiography emanates from 3-BrPA that is non-reactive or non-toxic, hence we further investigated whether the lack of toxicity is due to its interaction or alkylation with serum components. Analysis of serum proteins by 1D and 2D-gel electrophoretic autoradiography showed that 14C-BrPA selectively binds to peptides of molecular mass ~50-60 kDa. Mass spectrometry data suggested that 14C-BrPA could interact with alpha1-antitrypsin and a peptide of albuminoid-family. Our data indicate that selective interaction of 3-BrPA with serum proteins could contribute to the apparent lack of tissue-toxicity at the indicated close when the drug is given systematically in Sprague-Dawley rats.

  13. Radiocarbon Dating of the Human Eye Lens Crystallines Reveal Proteins without Carbon Turnover throughout Life

    Science.gov (United States)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen; Jacobsen, Christina; Heinemeier, Jan

    2008-01-01

    Background Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule) completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its 14C content from the atmospheric carbon dioxide. The 14C content of the lens proteins thus reflects the atmospheric content of 14C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the 14C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric 14C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating. Methodology/Principal Findings Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation. Conclusions/Significance Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the 14C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA). Potential targets may be nervous tissues in terms of senile or pre-senile degradation, as well as other highly

  14. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life.

    Directory of Open Access Journals (Sweden)

    Niels Lynnerup

    Full Text Available BACKGROUND: Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14C content from the atmospheric carbon dioxide. The (14C content of the lens proteins thus reflects the atmospheric content of (14C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the (14C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric (14C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating. METHODOLOGY/PRINCIPAL FINDINGS: Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation. CONCLUSIONS/SIGNIFICANCE: Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the (14C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA. Potential targets may be nervous tissues in terms of senile or pre

  15. Protein binding of glufosinate and factors affecting it revealed by an equilibrium dialysis technique.

    Science.gov (United States)

    Hori, Y; Koyama, K; Fujisawa, M; Nakajima, M; Shimada, K; Hirose, Y; Kohda, Y; Akuzawa, H

    2001-09-01

    We investigated the protein binding of glufosinate ammonium (GLF) and several factors affecting this binding using human serum albumin (HSA) and human volunteer serum under various conditions. The mean ratios of the free GLF (RFr-GLF) to 4% HSA were examined in the sera of patients described elsewhere at GLF levels from 1 microg/mL to 500 microg/mL; the range was found to be only from 0.80 to 0.88. Neither the incubation temperature nor buffers containing different chloride ion concentrations had any effect on the RFr-GLF to HSA. Moreover, the addition of heparin, glycoprotein-alpha1-acid (AAG), and sodium azide had no effect on the RFr-GLF. However, pH of the isotonic phosphate buffer and the addition of palmitic or oleic acid were seen to have an effect. In this study, the mean RFr-GLF to healthy human serum was 0.99. This high value was evidenced that GLF was rapidly excreted through the renal route.

  16. Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

    Directory of Open Access Journals (Sweden)

    Nicholas T. Ingolia

    2014-09-01

    Full Text Available Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs. Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

  17. Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

    Science.gov (United States)

    Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

    2011-12-01

    Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent

  18. Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

    Science.gov (United States)

    Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

    2015-01-01

    The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target.

  19. Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

    Science.gov (United States)

    Borin, Gustavo Pagotto; Sanchez, Camila Cristina; de Souza, Amanda Pereira; de Santana, Eliane Silva; de Souza, Aline Tieppo; Leme, Adriana Franco Paes; Squina, Fabio Marcio; Buckeridge, Marcos; Goldman, Gustavo Henrique; Oliveira, Juliana Velasco de Castro

    2015-01-01

    Background Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant. Results Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external

  20. Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass.

    Directory of Open Access Journals (Sweden)

    Gustavo Pagotto Borin

    Full Text Available Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses, must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external

  1. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    International Nuclear Information System (INIS)

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-01-01

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners

  2. Secretome of Aggregated Embryonic Stem Cell-Derived Mesenchymal Stem Cell Modulates the Release of Inflammatory Factors in Lipopolysaccharide-Induced Peripheral Blood Mononuclear Cells

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    Mohammadi Ghahhari, Nastaran; Maghsood, Faezeh; Jahandideh, Saeed; Lotfinia, Majid; Lak, Shirin; Johari, Behrooz; Azarnezhad, Asaad; Kadivar, Mehdi

    2018-07-01

    Bone marrow mesenchymal stem cells (BM-MSCs) have emerged as a potential therapy for various inflammatory diseases. Because of some limitations, several recent studies have suggested the use of embryonic stem cell-derived MSCs (ESC-MSCs) as an alternative for BM-MSCs. Some of the therapeutic effects of the ESC-MSCs are related to the secretion of a broad array of cytokines and growth factors, known as secretome. Harnessing this secretome for therapeutic applications requires the optimization of production of secretary molecules. It has been shown that aggregation of MSCs into 3D spheroids, as a preconditioning strategy, can enhance immunomodulatory potential of such cells. In this study, we investigated the effect of secretome derived from human ESC-MSCs (hESC-MSCs) spheroids on secretion of IL-1β, IL-10, and tumor necrosis factor α (TNF-α) from lipopolysaccharide (LPS)-induced peripheral blood mononuclear cells (PBMCs). In the present study, after immunophenotyping and considering mesodermal differentiation of hESC-MSCs, the cells were non-adherently grown to prepare 3D aggregates, and then conditioned medium or secretome was extracted from the cultures. Afterwards, the anti-inflammatory effects of the secretome were assessed in an in vitro model of inflammation. Results from this study showed that aggregate-prepared secretome from hESC-MSCs was able to significantly decrease the secretion of TNF-α (301.7 ± 5.906, p strategy to increase immunomodulatory characteristics of hESC-MSCs.

  3. What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

    Science.gov (United States)

    Ogorzalek Loo, Rachel R.; Lakshmanan, Rajeswari; Loo, Joseph A.

    2014-10-01

    Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range, and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate with those observed by ESI-MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase, the extent of charging. This region incorporates properties (e.g., basicities) intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging ("supercharging") such as m-NBA, sulfolane, and 3-nitrobenzonitrile increase analyte charge from "denaturing" and "native" solvent systems. It is suggested that additives' Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte's solution ionization, excess spray charge is bestowed on evaporating ions carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte).

  4. Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway

    Science.gov (United States)

    Murali, Rajmohan; Chandramohan, Raghu; Möller, Inga; Scholz, Simone L.; Berger, Michael; Huberman, Kety; Viale, Agnes; Pirun, Mono; Socci, Nicholas D.; Bouvier, Nancy; Bauer, Sebastian; Artl, Monika; Schilling, Bastian; Schimming, Tobias; Sucker, Antje; Schwindenhammer, Benjamin; Grabellus, Florian; Speicher, Michael R.; Schaller, Jörg; Hillen, Uwe; Schadendorf, Dirk; Mentzel, Thomas; Cheng, Donavan T.; Wiesner, Thomas; Griewank, Klaus G.

    2015-01-01

    Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas. PMID:26440310

  5. Proteomic profiling of human keratinocytes undergoing UVB-induced alternative differentiation reveals TRIpartite Motif Protein 29 as a survival factor.

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    Véronique Bertrand-Vallery

    Full Text Available BACKGROUND: Repeated exposures to UVB of human keratinocytes lacking functional p16(INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. METHODOLOGY/PRINCIPAL FINDINGS: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29. Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCdelta. CONCLUSIONS/SIGNIFICANCE: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.

  6. Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein.

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    Plchova, Helena; Hartung, Frank; Puchta, Holger

    2003-11-07

    The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.

  7. Rapid evolution of the sequences and gene repertoires of secreted proteins in bacteria.

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    Teresa Nogueira

    Full Text Available Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.

  8. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

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    Breitkopf, Susanne B.; Yang, Xuemei; Begley, Michael J.; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B.; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A.; Hong, Pengyu; Kontaridis, Maria I.; Cantley, Lewis C.; Perrimon, Norbert; Asara, John M.

    2016-02-01

    Using a series of immunoprecipitation (IP) - tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

  9. Mesenchymal Stem Cell Secretome: A Potential Tool for the Prevention of Muscle Degenerative Changes Associated With Chronic Rotator Cuff Tears.

    Science.gov (United States)

    Sevivas, Nuno; Teixeira, Fábio Gabriel; Portugal, Raquel; Araújo, Luís; Carriço, Luís Filipe; Ferreira, Nuno; Vieira da Silva, Manuel; Espregueira-Mendes, João; Anjo, Sandra; Manadas, Bruno; Sousa, Nuno; Salgado, António J

    2016-08-08

    Massive rotator cuff tears (MRCTs) are usually chronic lesions with pronounced degenerative changes, where advanced fatty degeneration and atrophy can make the tear irreparable. Human mesenchymal stem cells (hMSCs) secrete a range of growth factors and vesicular systems, known as secretome, that mediates regenerative processes in tissues undergoing degeneration. To study the effect of hMSC secretome on muscular degenerative changes and shoulder function on a rat MRCT model. Controlled laboratory study. A bilateral 2-tendon (supraspinatus and infraspinatus) section was performed to create an MRCT in a rat model. Forty-four Wistar-Han rats were randomly assigned to 6 groups: control group (sham surgery), lesion control group (MRCT), and 4 treated-lesion groups according to the site and periodicity of hMSC secretome injection: single local injection, multiple local injections, single systemic injection, and multiple systemic injections. Forelimb function was analyzed with the staircase test. Atrophy and fatty degeneration of the muscle were evaluated at 8 and 16 weeks after injury. A proteomic analysis was conducted to identify the molecules present in the hMSC secretome that can be associated with muscular degeneration prevention. When untreated for 8 weeks, the MRCT rats exhibited a significantly higher fat content (0.73% ± 0.19%) compared with rats treated with a single local injection (0.21% ± 0.04%; P muscle atrophy, 8 weeks after injury, only the single local injection group (0.0993% ± 0.0036%) presented a significantly higher muscle mass than that of the untreated MRCT group (0.0794% ± 0.0047%; P muscle regeneration, namely, pigment epithelium-derived factor and follistatin. The study data suggest that hMSC secretome effectively decreases the fatty degeneration and atrophy of the rotator cuff muscles. We describe a new approach for decreasing the characteristic muscle degeneration associated with chronic rotator cuff tears. This strategy is particularly

  10. Directed evolution and in silico analysis of reaction centre proteins reveal molecular signatures of photosynthesis adaptation to radiation pressure.

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    Giuseppina Rea

    2011-01-01

    Full Text Available Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues

  11. Systematic analysis of compositional order of proteins reveals new characteristics of biological functions and a universal correlate of macroevolution.

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    Erez Persi

    Full Text Available We present a novel analysis of compositional order (CO based on the occurrence of Frequent amino-acid Triplets (FTs that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between 'regularity', 'periodicity' and 'vocabulary', to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic 'innovation' at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces.

  12. Systematic Analysis of Compositional Order of Proteins Reveals New Characteristics of Biological Functions and a Universal Correlate of Macroevolution

    Science.gov (United States)

    Persi, Erez; Horn, David

    2013-01-01

    We present a novel analysis of compositional order (CO) based on the occurrence of Frequent amino-acid Triplets (FTs) that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between ‘regularity’, ‘periodicity’ and ‘vocabulary’, to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT) in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic ‘innovation’ at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces. PMID:24278003

  13. Recent Advances in In Vitro Fertilization: Proteomics, Secretomics, Metabolomics and In Vitro Maturation

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    Ercan Baştu

    2013-03-01

    Full Text Available Since its first successful result in 1978, clinicians and researchers have been working on increasing the efficiency and safety of in vitro fertilization (IVF. As a result of advances in technology and understanding of human reproduction, IVF success rates have increased while high-order multiple pregnancy (triplets and more rates have decreased. On the other, there is opportunity for further improvement as many couples still face ‘unexplained infertility’ and high rates of twin pregnancies. Latest technologic and scientific improvements in IVF are promising. The aim of this review is to present the latest advances in the fields of proteomics, secretomics, metabolomics and oocyte culture, how they can potentially improve embryo selection and in vitro maturation (IVM and subsequently their possible impact on the safety and efficacy of IVF.

  14. Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of cereal yellow dwarf virus.

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    Cecilia Tamborindeguy

    Full Text Available Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV. The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S. graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate

  15. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

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    Olivier Poupel

    Full Text Available The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their

  16. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  17. Interaction study of rice stripe virus proteins reveals a region of the nucleocapsid protein (NP) required for NP self-interaction and nuclear localization.

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    Lian, Sen; Cho, Won Kyong; Jo, Yeonhwa; Kim, Sang-Min; Kim, Kook-Hyung

    2014-04-01

    Rice stripe virus (RSV), which belongs to the genus Tenuivirus, is an emergent virus problem. The RSV genome is composed of four single-strand RNAs (RNA1-RNA4) and encodes seven proteins. We investigated interactions between six of the RSV proteins by yeast-two hybrid (Y2H) assay in vitro and by bimolecular fluorescence complementation (BiFC) in planta. Y2H identified self-interaction of the nucleocapsid protein (NP) and NS3, while BiFC revealed self-interaction of NP, NS3, and NCP. To identify regions(s) and/or crucial amino acid (aa) residues required for NP self-interaction, we generated various truncated and aa substitution mutants. Y2H assay showed that the N-terminal region of NP (aa 1-56) is necessary for NP self-interaction. Further analysis with substitution mutants demonstrated that additional aa residues located at 42-47 affected their interaction with full-length NP. These results indicate that the N-terminal region (aa 1-36 and 42-47) is required for NP self-interaction. BiFC and co-localization studies showed that the region required for NP self-interaction is also required for NP localization at the nucleus. Overall, our results indicate that the N-terminal region (aa 1-47) of the NP is important for NP self-interaction and that six aa residues (42-47) are essential for both NP self-interaction and nuclear localization. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis

    Science.gov (United States)

    Kirkpatrick, Christine L.; Parsley, Nicole C.; Bartges, Tessa E.; Cooke, Madeline E.; Evans, Wilaysha S.; Heil, Lilian R.; Smith, Thomas J.; Hicks, Leslie M.

    2018-05-01

    Fungal secondary metabolites represent a rich and largely untapped source for bioactive molecules, including peptides with substantial structural diversity and pharmacological potential. As methods proceed to take a deep dive into fungal genomes, complimentary methods to identify bioactive components are required to keep pace with the expanding fungal repertoire. We developed PepSAVI-MS to expedite the search for natural product bioactive peptides and herein demonstrate proof-of-principle applicability of the pipeline for the discovery of bioactive peptides from fungal secretomes via identification of the antifungal killer toxin KP4 from Ustilago maydis P4. This work opens the door to investigating microbial secretomes with a new lens, and could have broad applications across human health, agriculture, and food safety. [Figure not available: see fulltext.

  19. "Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

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    Tischer, Alexander; Machha, Venkata R; Rösgen, Jörg; Auton, Matthew

    2018-02-19

    Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how ΔH and the urea m-value interconvert through the slope of c m versus T, (∂cm/∂T)=ΔH/(mT). This relationship permits the calculation of ΔH at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from ΔH obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of ΔH and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall

  20. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    Science.gov (United States)

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  1. Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

    Directory of Open Access Journals (Sweden)

    Patricia eNAGNAN-LE MEILLOUR

    2014-12-01

    Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

  2. Use of a molecular genetic platform technology to produce human Wnt proteins reveals distinct local and distal signaling abilities.

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    Jennifer L Green

    Full Text Available Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.

  3. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

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    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  4. Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

    Science.gov (United States)

    De Souza, Colin P; Hashmi, Shahr B; Osmani, Aysha H; Osmani, Stephen A

    2014-01-01

    Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients

  5. Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

    Science.gov (United States)

    Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

    2014-12-01

    Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. PROS-1/Prospero Is a Major Regulator of the Glia-Specific Secretome Controlling Sensory-Neuron Shape and Function in C. elegans.

    Science.gov (United States)

    Wallace, Sean W; Singhvi, Aakanksha; Liang, Yupu; Lu, Yun; Shaham, Shai

    2016-04-19

    Sensory neurons are an animal's gateway to the world, and their receptive endings, the sites of sensory signal transduction, are often associated with glia. Although glia are known to promote sensory-neuron functions, the molecular bases of these interactions are poorly explored. Here, we describe a post-developmental glial role for the PROS-1/Prospero/PROX1 homeodomain protein in sensory-neuron function in C. elegans. Using glia expression profiling, we demonstrate that, unlike previously characterized cell fate roles, PROS-1 functions post-embryonically to control sense-organ glia-specific secretome expression. PROS-1 functions cell autonomously to regulate glial secretion and membrane structure, and non-cell autonomously to control the shape and function of the receptive endings of sensory neurons. Known glial genes controlling sensory-neuron function are PROS-1 targets, and we identify additional PROS-1-dependent genes required for neuron attributes. Drosophila Prospero and vertebrate PROX1 are expressed in post-mitotic sense-organ glia and astrocytes, suggesting conserved roles for this class of transcription factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Excreted/Secreted Proteins from Trypanosome Procyclic Strains

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    Celestine Michelle Atyame Nten

    2010-01-01

    Full Text Available Trypanosoma secretome was shown to be involved in parasite virulence and is suspected of interfering in parasite life-cycle steps such as establishment in the Glossina midgut, metacyclogenesis. Therefore, we attempted to identify the proteins secreted by procyclic strains of T. brucei gambiense and T. brucei brucei, responsible for human and animal trypanosomiasis, respectively. Using mass spectrometry, 427 and 483 nonredundant proteins were characterized in T. brucei brucei and T. brucei gambiense secretomes, respectively; 35% and 42% of the corresponding secretome proteins were specifically secreted by T. brucei brucei and T. brucei gambiense, respectively, while 279 proteins were common to both subspecies. The proteins were assigned to 12 functional classes. Special attention was paid to the most abundant proteases (14 families because of their potential implication in the infection process and nutrient supply. The presence of proteins usually secreted via an exosome pathway suggests that this type of process is involved in trypanosome ESP secretion. The overall results provide leads for further research to develop novel tools for blocking trypanosome transmission.

  8. Molecular Chemical Structure of Barley Proteins Revealed by Ultra-Spatially Resolved Synchrotron Light Sourced FTIR Microspectroscopy: Comparison of Barley Varieties

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Barley protein structure affects the barley quality, fermentation, and degradation behavior in both humans and animals among other factors such as protein matrix. Publications show various biological differences among barley varieties such as Valier and Harrington, which have significantly different degradation behaviors. The objectives of this study were to reveal the molecular structure of barley protein, comparing various varieties (Dolly, Valier, Harrington, LP955, AC Metcalfe, and Sisler), and quantify protein structure profiles using Gaussian and Lorentzian methods of multi-component peak modeling by using the ultra-spatially resolved synchrotron light sourced Fourier transform infrared microspectroscopy (SFTIRM). The items of the protein molecular structure revealed included protein structure α-helices, β-sheets, and others such as β-turns and random coils. The experiment was performed at the National Synchrotron Light Source in Brookhaven National Laboratory (BNL, US Department of Energy, NY). The results showed that with the SFTIRM, the molecular structure of barley protein could be revealed. Barley protein structures exhibited significant differences among the varieties in terms of proportion and ratio of model-fitted α-helices, β-sheets, and others. By using multi-component peaks modeling at protein amide I region of 1710-1576 cm -1 , the results show that barley protein consisted of approximately 18-34% of α-helices, 14-25% of β-sheets, and 44-69% others. AC Metcalfe, Sisler, and LP955 consisted of higher (P 0.05). The ratio of α-helices to others (0.3 to 1.0, P < 0.05) and that of β-sheets to others (0.2 to 0.8, P < 0.05) were different among the barley varieties. It needs to be pointed out that using a multi-peak modeling for protein structure analysis is only for making relative estimates and not exact determinations and only for the comparison purpose between varieties. The principal component analysis showed that protein amide I Fourier

  9. Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.

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    Myriam A Badr

    Full Text Available Cardiac troponin C (cTnC is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP and an acceptor fluorescent protein (YFP. The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+ conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

  10. Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

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    Olgun eGuvench

    2015-06-01

    Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

  11. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

    NARCIS (Netherlands)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-01-01

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the

  12. Dissection of an old protein reveals a novel application: domain D of Staphylococcus aureus Protein A (sSpAD as a secretion - tag

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    Paal Michael

    2010-11-01

    Full Text Available Abstract Background Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous. Results We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation. The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD. Conclusions In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.

  13. Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.

    Science.gov (United States)

    Bekliz, Meriem; Azza, Said; Seligmann, Hervé; Decloquement, Philippe; Raoult, Didier; La Scola, Bernard

    2018-05-15

    The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of

  14. Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements.

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    Meghana Deepak Shirke

    Full Text Available Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck, finger millet (leaf and neck, foxtail millet (leaf and buffel grass (leaf. Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors.

  15. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

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    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  16. Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

    Science.gov (United States)

    Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

    2017-07-01

    The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination

    International Nuclear Information System (INIS)

    Mizuno, Hideaki; Mal, Tapas Kumar; Waelchli, Markus; Fukano, Takashi; Ikura, Mitsuhiko; Miyawaki, Atsushi

    2010-01-01

    Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of 13 C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C α and C β of Y63 in the chromophore, the β-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of β-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.

  18. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    OpenAIRE

    Herranz, M. Carmen; Sánchez Navarro, Jesús A.; Saurí Peris, Ana; Mingarro Muñoz, Ismael; Pallás Benet, Vicente

    2005-01-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive c...

  19. Systematic differences in signal emitting and receiving revealed by PageRank analysis of a human protein interactome.

    Directory of Open Access Journals (Sweden)

    Donglei Du

    Full Text Available Most protein PageRank studies do not use signal flow direction information in protein interactions because this information was not readily available in large protein databases until recently. Therefore, four questions have yet to be answered: A What is the general difference between signal emitting and receiving in a protein interactome? B Which proteins are among the top ranked in directional ranking? C Are high ranked proteins more evolutionarily conserved than low ranked ones? D Do proteins with similar ranking tend to have similar subcellular locations? In this study, we address these questions using the forward, reverse, and non-directional PageRank approaches to rank an information-directional network of human proteins and study their evolutionary conservation. The forward ranking gives credit to information receivers, reverse ranking to information emitters, and non-directional ranking mainly to the number of interactions. The protein lists generated by the forward and non-directional rankings are highly correlated, but those by the reverse and non-directional rankings are not. The results suggest that the signal emitting/receiving system is characterized by key-emittings and relatively even receivings in the human protein interactome. Signaling pathway proteins are frequent in top ranked ones. Eight proteins are both informational top emitters and top receivers. Top ranked proteins, except a few species-related novel-function ones, are evolutionarily well conserved. Protein-subunit ranking position reflects subunit function. These results demonstrate the usefulness of different PageRank approaches in characterizing protein networks and provide insights to protein interaction in the cell.

  20. Systematic differences in signal emitting and receiving revealed by PageRank analysis of a human protein interactome.

    Science.gov (United States)

    Du, Donglei; Lee, Connie F; Li, Xiu-Qing

    2012-01-01

    Most protein PageRank studies do not use signal flow direction information in protein interactions because this information was not readily available in large protein databases until recently. Therefore, four questions have yet to be answered: A) What is the general difference between signal emitting and receiving in a protein interactome? B) Which proteins are among the top ranked in directional ranking? C) Are high ranked proteins more evolutionarily conserved than low ranked ones? D) Do proteins with similar ranking tend to have similar subcellular locations? In this study, we address these questions using the forward, reverse, and non-directional PageRank approaches to rank an information-directional network of human proteins and study their evolutionary conservation. The forward ranking gives credit to information receivers, reverse ranking to information emitters, and non-directional ranking mainly to the number of interactions. The protein lists generated by the forward and non-directional rankings are highly correlated, but those by the reverse and non-directional rankings are not. The results suggest that the signal emitting/receiving system is characterized by key-emittings and relatively even receivings in the human protein interactome. Signaling pathway proteins are frequent in top ranked ones. Eight proteins are both informational top emitters and top receivers. Top ranked proteins, except a few species-related novel-function ones, are evolutionarily well conserved. Protein-subunit ranking position reflects subunit function. These results demonstrate the usefulness of different PageRank approaches in characterizing protein networks and provide insights to protein interaction in the cell.

  1. Analysis of Secreted Proteins Using SILAC

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Blagoev, Blagoy; Kratchmarova, Irina

    2014-01-01

    Secreted proteins serve a crucial role in the communication between cells, tissues, and organs. Proteins released to the extracellular environment exert their function either locally or at distant points of the organism. Proteins are secreted in a highly dynamic fashion by cells and tissues...... in the body responding to the stimuli and requirements presented by the extracellular milieu. Characterization of secretomes derived from various cell types has been performed using different quantitative mass spectrometry-based proteomics strategies, several of them taking advantage of labeling with stable...

  2. Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected

    Energy Technology Data Exchange (ETDEWEB)

    Sushko, Oleksandr; Dubrovka, Rostyslav; Donnan, Robert S., E-mail: r.donnan@qmul.ac.uk [School of Electronic Engineering and Computer Science, Queen Mary University of London, Mile End Road, London E1 4NS (United Kingdom)

    2015-02-07

    The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 Å, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity.

  3. Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected

    International Nuclear Information System (INIS)

    Sushko, Oleksandr; Dubrovka, Rostyslav; Donnan, Robert S.

    2015-01-01

    The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 Å, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity

  4. Quantitative proteome and phosphoproteome analyses of Streptomyces coelicolor reveal proteins and phosphoproteins modulating differentiation and secondary metabolism

    DEFF Research Database (Denmark)

    Rioseras, Beatriz; Sliaha, Pavel V; Gorshkov, Vladimir

    2018-01-01

    identified and quantified 3461 proteins corresponding to 44.3% of the S. coelicolor proteome across three developmental stages: vegetative hypha (MI); secondary metabolite producing hyphae (MII); and sporulating hyphae. A total of 1350 proteins exhibited more than 2-fold expression changes during....../Thr/Tyr kinases, making this genus an outstanding model for the study of bacterial protein phosphorylation events. We used mass spectrometry based quantitative proteomics and phosphoproteomics to characterize bacterial differentiation and activation of secondary metabolism of Streptomyces coelicolor. We...... the bacterial differentiation process. These proteins include 136 regulators (transcriptional regulators, transducers, Ser/Thr/Tyr kinases, signalling proteins), as well as 542 putative proteins with no clear homology to known proteins which are likely to play a role in differentiation and secondary metabolism...

  5. Protein expression profiling of inflammatory mediators in human temporal lobe epilepsy reveals co-activation of multiple chemokines and cytokines

    Directory of Open Access Journals (Sweden)

    Kan Anne A

    2012-08-01

    Full Text Available Abstract Mesial temporal lobe epilepsy (mTLE is a chronic and often treatment-refractory brain disorder characterized by recurrent seizures originating from the hippocampus. The pathogenic mechanisms underlying mTLE remain largely unknown. Recent clinical and experimental evidence supports a role of various inflammatory mediators in mTLE. Here, we performed protein expression profiling of 40 inflammatory mediators in surgical resection material from mTLE patients with and without hippocampal sclerosis, and autopsy controls using a multiplex bead-based immunoassay. In mTLE patients we identified 21 upregulated inflammatory mediators, including 10 cytokines and 7 chemokines. Many of these upregulated mediators have not previously been implicated in mTLE (for example, CCL22, IL-7 and IL-25. Comparing the three patient groups, two main hippocampal expression patterns could be distinguished, pattern I (for example, IL-10 and IL-25 showing increased expression in mTLE + HS patients compared to mTLE-HS and controls, and pattern II (for example, CCL4 and IL-7 showing increased expression in both mTLE groups compared to controls. Upregulation of a subset of inflammatory mediators (for example, IL-25 and IL-7 could not only be detected in the hippocampus of mTLE patients, but also in the neocortex. Principle component analysis was used to cluster the inflammatory mediators into several components. Follow-up analyses of the identified components revealed that the three patient groups could be discriminated based on their unique expression profiles. Immunocytochemistry showed that IL-25 IR (pattern I and CCL4 IR (pattern II were localized in astrocytes and microglia, whereas IL-25 IR was also detected in neurons. Our data shows co-activation of multiple inflammatory mediators in hippocampus and neocortex of mTLE patients, indicating activation of multiple pro- and anti-epileptogenic immune pathways in this disease.

  6. Analysis of the embryo proteome of sycamore (Acer pseudoplatanus L.) seeds reveals a distinct class of proteins regulating dormancy release.

    Science.gov (United States)

    Pawłowski, Tomasz Andrzej; Staszak, Aleksandra Maria

    2016-05-20

    Acer pseudoplatanus seeds are characterized by a deep physiological embryo dormancy that requires a few weeks of cold stratification in order to promote germination. Understanding the function of proteins and their related metabolic pathways, in conjunction with the plant hormones implicated in the breaking of seed dormancy, would expand our knowledge pertaining to this process. In this study, a proteomic approach was used to analyze the changes occurring in seeds in response to cold stratification, which leads to dormancy release. In addition, the involvement of abscisic (ABA) and gibberellic acids (GA) was also examined. Fifty-three proteins showing significant changes were identified by mass spectrometry. An effect of ABA on protein variation was observed at the beginning of stratification, while the influence of GA on protein abundance was observed during the middle phase of stratification. The majority of proteins associated with dormancy breaking in the presence of only water, and also ABA or GA, were classified as being involved in metabolism and genetic information processing. For metabolic-related proteins, the effect of ABA on protein abundance was stimulatory for half of the proteins and inhibitory for half of the proteins. On the other hand, the effect on genetic information processing related proteins was stimulatory. GA was found to upregulate both metabolic-related and genetic information processing-related proteins. While seed dormancy breaking depends on proteins involved in a variety of processes, proteins associated with methionine metabolism (adenosine kinase, methionine synthase) and glycine-rich RNA binding proteins appear to be of particular importance. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    Science.gov (United States)

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-05

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

  8. Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

    Science.gov (United States)

    Kandhavelu, Jeyalakshmi; Demonte, Naveen Luke; Namperumalsamy, Venkatesh Prajna; Prajna, Lalitha; Thangavel, Chitra; Jayapal, Jeya Maheshwari; Kuppamuthu, Dharmalingam

    2017-01-30

    Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense

  9. Identifying Bacterial Immune Evasion Proteins Using Phage Display.

    Science.gov (United States)

    Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

    2017-01-01

    Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

  10. A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

    Science.gov (United States)

    Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

    2008-10-01

    Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

  11. The structure of mAG, a monomeric mutant of the green fluorescent protein Azami-Green, reveals the structural basis of its stable green emission

    International Nuclear Information System (INIS)

    Ebisawa, Tatsuki; Yamamura, Akihiro; Kameda, Yasuhiro; Hayakawa, Kou; Nagata, Koji; Tanokura, Masaru

    2010-01-01

    The crystal structure of a monomeric mutant of Azami-Green (mAG) from G. fascicularis was determined at 2.2 Å resolution. Monomeric Azami-Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green-emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino-acid sequences. mAG is more similar in its amino-acid sequence to four fluorescent proteins: Dendra2 (a green-to-red irreversibly photoconverting fluorescent protein), Dronpa (a bright-and-dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green-emitting fluorescent protein) and Kaede (another green-to-red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62-Tyr63-Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants

  12. The unique architecture and function of cellulose-interacting proteins in oomycetes revealed by genomic and structural analyses

    Directory of Open Access Journals (Sweden)

    Larroque Mathieu

    2012-11-01

    Full Text Available Abstract Background Oomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1. Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL, a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity. Results To extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs. Conclusions This study

  13. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    Directory of Open Access Journals (Sweden)

    Allison Marie Barbaglia

    2016-04-01

    Full Text Available Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho- lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012. Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I a putative GDSL-motif lipase (II a PIG-P-like protein, with a possible receptor-like function; (III and PLAFP (phloem lipid-associated family protein, a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH, which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while

  14. Structural and functional implications in the eubacterial ribosome as revealed by protein-rRNA and antibiotic contact sites.

    Science.gov (United States)

    Wittmann-Liebold, B; Uhlein, M; Urlaub, H; Müller, E C; Otto, A; Bischof, O

    1995-01-01

    Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2-iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at the peptide level in several proteins that were found to constitute the antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, AND L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA-peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 with a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.

  15. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

    Science.gov (United States)

    Haarmeyer, Carolyn N; Smith, Matthew D; Chundawat, Shishir P S; Sammond, Deanne; Whitehead, Timothy A

    2017-04-01

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is t