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Sample records for domain-containing protein hic

  1. UBA domain containing proteins in fission yeast

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Semple, Colin A M; Ponting, Chris P

    2003-01-01

    characterised on both the functional and structural levels. One example of a widespread ubiquitin binding module is the ubiquitin associated (UBA) domain. Here, we discuss the approximately 15 UBA domain containing proteins encoded in the relatively small genome of the fission yeast Schizosaccharomyces pombe...

  2. Structure function relations in PDZ-domain-containing proteins ...

    Indian Academy of Sciences (India)

    G P Manjunath

    2017-12-30

    Dec 30, 2017 ... associated with autism spectrum disorders (Zeng et al. 2016a;. Monteiro and Feng 2017). Further, there are reports sup- porting the role of PDZ proteins in neuronal remodelling via the regulation of microtubule dynamics (Sweet et al. 2011;. Chen et al. 2014). Not all PDZ-domain-containing proteins.

  3. HIC

    DEFF Research Database (Denmark)

    Christiansen, Henning

    2016-01-01

    HIC is an interactive system for image classification system on supervised machine learning written in Processing, processing.org. It has been developed specifically for the course Artificial Intelligence in Interactive Systems, given at Roskilde's Humanities-Technology Bachelor studies. This web...

  4. Structure function relations in PDZ-domain-containing proteins ...

    Indian Academy of Sciences (India)

    G P Manjunath

    2017-12-30

    Dec 30, 2017 ... cost to the organism by the appearance of functionally redundant ... fashion, making them both robust as well as highly responsive to the ...... Trends Cell Biol. 10. 32–38. Basdevant N, Weinstein H and Ceruso M 2006 Thermodynamic basis for promiscuity and selectivity in protein–protein interac- tions: PDZ ...

  5. Understanding the role of BAR and SH3 domain-containing proteins in fungi

    NARCIS (Netherlands)

    Gkourtsa, A.

    2017-01-01

    This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein

  6. α/β-hydrolase domain containing protein 15 (ABHD15--an adipogenic protein protecting from apoptosis.

    Directory of Open Access Journals (Sweden)

    Evelyn Walenta

    Full Text Available Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15 is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ, the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  7. A novel PAN/apple domain-containing protein from Toxoplasma gondii: characterization and receptor identification.

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    Haiyan Gong

    Full Text Available Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677 is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG interaction. Chondroitin sulfate (CS, a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

  8. Paragonimus westermani: identification and characterization of the fasciclin I domain-containing protein.

    Science.gov (United States)

    Song, Su-Min; Shin, Jong-Won; de Guzman, Jefferson V; Kim, Jin; Yu, Hak-Sun; Jha, Bijay Kumar; Kong, Hyun-Hee; Hong, Yeonchul; Chung, Dong-Il

    2010-06-01

    Paragonimus westermani is a trematode parasite that causes inflammatory lung disease as well as systemic infections in carnivorous mammals. The interaction of the parasite with host cells and paired worms is initiated by adhesion and plays an important role in parasite proliferation and differentiation. In this study, we isolated a cDNA encoding a P. westermani fasciclin I domain-containing protein (Pwfas-I). The fasiclin-I domain is suggested to be involved in cell adhesion, migration, and differentiation. Immunohistochemical analysis of P. westermani adult worms with polyclonal anti-Pwfas-I serum revealed immunoreactivity in the egg shells and the cells lining the sub-tegumental layer of adult worm throughout the contact regions of the cyst wall and paired worms. Using cell adhesion and spreading assays, we showed that Pwfas-I supports cell adhesion and spreading. Furthermore, we determined that the alphanubeta5 integrin was a functional receptor for the Pwfas-I. Taken together, these results suggest that Pwfas-I may be functional for the modulation of cell adhesion via binding with alphanubeta5 integrin in the extracellular matrix of Paragonimus. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  9. Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension.

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    Alan Y Deng

    Full Text Available Multiple quantitative trait loci (QTLs for blood pressure (BP have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1/activator of G protein signaling 8 (Ags8 to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5' Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP 'indirectly' by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy.

  10. The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

    Science.gov (United States)

    Choo, Jocelyn M; Cheung, Jackie K; Wisniewski, Jessica A; Steer, David L; Bulach, Dieter M; Hiscox, Thomas J; Chakravorty, Anjana; Smith, A Ian; Gell, David A; Rood, Julian I; Awad, Milena M

    2016-01-01

    The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

  11. Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

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    Žárský Viktor

    2008-04-01

    Full Text Available Abstract Background Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures. Results The Rho GTPase-binding domain (GBD/FH3 reported from opisthokont and Dictyostelium formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm formins that define a specific branch (Class III of the formin family. Conclusion We propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms.

  12. Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

    Science.gov (United States)

    Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

    2016-07-01

    Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. Copyright © 2016 by the Genetics Society of America.

  13. Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

    Science.gov (United States)

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Habermeyer, Johanna; Schwertz, Hansjörg; Hammerschmidt, Sven

    2017-04-07

    Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Tudor Domain Containing Protein TDRD12 Expresses at the Acrosome of Spermatids in Mouse Testis

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    Min Kim

    2016-07-01

    Full Text Available Tdrd12 is one of tudor domain containing (Tdrd family members. However, the expression pattern of Tdrd12 has not been well studied. To compare the expression levels of Tdrd12 in various tissues, real time-polymerase chain reaction was performed using total RNAs from liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Tdrd12 mRNA was highly expressed in testis. Antibody against mouse TDRD12 were generated using amino acid residues SQRPNEKPLRLTEKKDC of TDRD12 to investigate TDRD12 localization in testis. Immunostaining assay shows that TDRD12 is mainly localized at the spermatid in the seminiferous tubules of adult testes. During postnatal development, TDRD12 is differentially expressed. TDRD12 was detected in early spermatocytes at 2 weeks and TDRD12 was localized at acrosome of the round spermatids. TDRD12 expression was not co-localized with TDRD1 which is an important component of piRNA pathway in germ cells. Our results indicate that TDRD12 may play an important role in spermatids and function as a regulator of spermatogenesis in dependent of TDRD1.

  15. The PAS Domain-Containing Protein HeuR Regulates Heme Uptake in Campylobacter jejuni

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    Jeremiah G. Johnson

    2016-11-01

    Full Text Available Campylobacter jejuni is a leading cause of bacterially derived gastroenteritis. A previous mutant screen demonstrated that the heme uptake system (Chu is required for full colonization of the chicken gastrointestinal tract. Subsequent work identified a PAS domain-containing regulator, termed HeuR, as being required for chicken colonization. Here we confirm that both the heme uptake system and HeuR are required for full chicken gastrointestinal tract colonization, with the heuR mutant being particularly affected during competition with wild-type C. jejuni. Transcriptomic analysis identified the chu genes—and those encoding other iron uptake systems—as regulatory targets of HeuR. Purified HeuR bound the chuZA promoter region in electrophoretic mobility shift assays. Consistent with a role for HeuR in chu expression, heuR mutants were unable to efficiently use heme as a source of iron under iron-limiting conditions, and mutants exhibited decreased levels of cell-associated iron by mass spectrometry. Finally, we demonstrate that an heuR mutant of C. jejuni is resistant to hydrogen peroxide and that this resistance correlates to elevated levels of catalase activity. These results indicate that HeuR directly and positively regulates iron acquisition from heme and negatively impacts catalase activity by an as yet unidentified mechanism in C. jejuni.

  16. Functional analysis of Tudor-domain-containing proteins in the zebrafish germline

    NARCIS (Netherlands)

    Huang, H.Y.

    2012-01-01

    The Argonaute protein family consists of two distinct sub-clades: AGO and PIWI proteins. AGO proteins can form RNA induced silencing complex (RISC) and are responsible for RNAi and miRNA pathways. PIWI proteins are mostly expressed in the germline and together with specific small RNA groups, named

  17. A Novel Matrix Protein Hic31 from the Prismatic Layer of Hyriopsis Cumingii Displays a Collagen-Like Structure.

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    Xiaojun Liu

    Full Text Available In this study, we clone and characterize a novel matrix protein, hic31, from the mantle of Hyriopsis cumingii. The amino acid composition of hic31 consists of a high proportion of Glycine residues (26.67%. Tissue expression detection by RT-PCR indicates that hic31 is expressed specifically at the mantle edge. In situ hybridization results reveals strong signals from the dorsal epithelial cells of the outer fold at the mantle edge, and weak signals from inner epithelial cells of the same fold, indicating that hic31 is a prismatic-layer matrix protein. Although BLASTP results identify no shared homology with other shell-matrix proteins or any other known proteins, the hic31 tertiary structure is similar to that of collagen I, alpha 1 and alpha 2. It has been well proved that collagen forms the basic organic frameworks in way of collagen fibrils and minerals present within or outside of these fibrils. Therefore, hic31 might be a framework-matrix protein involved in the prismatic-layer biomineralization. Besides, the gene expression of hic31 increase in the early stages of pearl sac development, indicating that hic31 may play important roles in biomineralization of the pearl prismatic layer.

  18. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

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    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  19. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-03-14

    Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  20. Chemical shift assignments of RHE_RS02845, a NTF2-like domain-containing protein from Rhizobium etli.

    Science.gov (United States)

    Li, Tao; Li, Shuangli; Liang, Chunjie; Zhu, Jiang; Liu, Maili; Yang, Yunhuang

    2018-03-23

    The nuclear transport factor 2 (NTF2) like superfamily includes members of the NTF2 family, delta-5-3-ketosteroid isomerases, and the beta subunit of ring hydroxygenases. This family plays important roles in both eukaryotic and prokaryotic cells, and is taken as a classic example of divergent evolution because proteins in this family exhibit diverse biological functions, although share common structural features. We cloned the gene RHE_RS02845 encoding a predicted NTF2-like domain-containing protein in Rhizobium etli, and prepared U- 13 C/ 15 N-labeled protein samples for its three-dimensional NMR structural determination. Here, chemical shift assignments for both backbone and side-chain atoms are reported, which is prerequisite for further structural calculation and functional research using NMR spectroscopy.

  1. Over-Expression of Rice CBS Domain Containing Protein, OsCBSX3, Confers Rice Resistance to Magnaporthe oryzae Inoculation

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    Shaoling Mou

    2015-07-01

    Full Text Available Cystathionine β-synthase (CBS domain containing proteins (CDCPs constitute a big family in plants and some members in this family have been implicated in a variety of biological processes, but the precise functions and the underlying mechanism of the majority of this family in plant immunity remain to be elucidated. In the present study, a CBS domain containing protein gene, OsCBSX3, is functionally characterized in rice resistance against Magnaporthe oryzae (M. oryzae. By quantitative real-time PCR, transcripts of OsCBSX3 are up-regulated significantly by inoculation of M. oryzae and the exogenously applied salicylic acid (SA and methyl jasmonate (MeJA. OsCBSX3 is exclusively localized to the plasma membrane by transient expression of OsCBSX3 fused to green fluorescent protein (GFP through approach of Agrobacterium infiltration in Nicotiana benthamiana leaves. The plants of homozygous T3 transgenic rice lines of over-expressing OsCBSX3 exhibit significant enhanced resistance to M. oryzae inoculation, manifested by decreased disease symptoms, and inhibition of pathogen growth detected in DNA. Consistently, the over-expression of OsCBSX3 enhances the transcript levels of immunity associated marker genes including PR1a, PR1b, PR5, AOS2, PAL, NH1, and OsWRKY13 in plants inoculated with M. oryzae. These results suggest that OsCBSX3 acts as a positive regulator in resistance of rice to M. oryzae regulated by SA and JA-mediated signaling pathways synergistically.

  2. Activation and Role of NACHT, LRR, and PYD Domains-Containing Protein 3 Inflammasome in RNA Viral Infection

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    Junyang Yu

    2017-10-01

    Full Text Available NACHT, LRR, and PYD domains-containing protein 3 (NLRP3 inflammasome activation and effects during ribonucleic acid (RNA viral infection are the focus of a wide range of research currently. Both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the NLRP3 inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. However, aberrant activation of the NLRP3 inflammasome can instead support viral pathogenesis and promote disease progression. Here, we summarize and expound upon the recent literature describing the molecular mechanisms underlying the activation and effects of the NLRP3 inflammasome in RNA viral infection to highlight how it provides protection against RNA viral infection.

  3. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

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    Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

    2005-11-21

    It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

  4. A START-domain-containing protein is a novel marker of nervous system components of the sea cucumber Holothuria glaberrima.

    Science.gov (United States)

    Rosado-Olivieri, Edwin A; Ramos-Ortiz, Gibram A; Hernández-Pasos, Josué; Díaz-Balzac, Carlos A; Vázquez-Rosa, Edwin; Valentín-Tirado, Griselle; Vega, Irving E; García-Arrarás, José E

    2017-12-01

    One of the main challenges faced by investigators studying the nervous system of members of the phylum Echinodermata is the lack of markers to identify nerve cells and plexi. Previous studies have utilized an antibody, RN1, that labels most of the nervous system structures of the sea cucumber Holothuria glaberrima and other echinoderms. However, the antigen recognized by RN1 remained unknown. In the present work, the antigen has been characterized by immunoprecipitation, tandem mass spectrometry, and cDNA cloning. The RN1 antigen contains a START lipid-binding domain found in Steroidogenic Acute Regulatory (StAR) proteins and other lipid-binding proteins. Phylogenetic tree assembly showed that the START domain is highly conserved among echinoderms. We have named this antigen HgSTARD10 for its high sequence similarity to the vertebrate orthologs. Gene and protein expression analyses revealed an abundance of HgSTARD10 in most H. glaberrima tissues including radial nerve, intestine, muscle, esophagus, mesentery, hemal system, gonads and respiratory tree. Molecular cloning of HgSTARD10, consequent protein expression and polyclonal antibody production revealed the STARD10 ortholog as the antigen recognized by the RN1 antibody. Further characterization into this START domain-containing protein will provide important insights for the biochemistry, physiology and evolution of deuterostomes. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Expression of WW domain-containing protein 2 is correlated with pathological grade and recurrence of glioma.

    Science.gov (United States)

    Liang, Jun; Qi, Wei-Feng; Xie, Shao; Wang, Wei-Feng; Zhang, Xian-Li; Zhou, Xiu-Ping; Hu, Jin-Xia; Yu, Ru-Tong

    2017-01-01

    WW domain-containing protein 2 (WWP2) is an E3 ubiquitin ligase, which belongs to the NEDD4-like protein family. Recently, it is reported to play a key role in tumorigenesis and development of tumors such as prostate and lung cancer. However, there has been not related report on glioma until now. The aim of this study is to detect the expression of WWP2 and analyze its correlation to the pathological grade and tumor recurrence in patients with glioma. Western blot and immunohistochemistry were separately used to detect the expression of WWP2 protein in 31 brain glioma tissue samples and 80 brain glioma paraffin specimens. The method of Kaplan-Meier was used to analyze the correlation between the WWP2 expression and glioma recurrence. The protein expression level of WWP2 in glioma tissue was significantly higher than that in nontumorous brain tissue (P level of WWP2 in high-grade glioma (Grade III-IV) was significantly higher than that in low-grade glioma (Grade I-II) (P study suggests that WWP2 may play a role in the genesis and development of glioma; it may be a potential biomarker to predict pathological grade and tumor recurrence in patients with glioma.

  6. A double WAP domain-containing protein Es-DWD1 from Eriocheir sinensis exhibits antimicrobial and proteinase inhibitory activities.

    Directory of Open Access Journals (Sweden)

    Shuang Li

    Full Text Available Whey acidic proteins (WAP belong to a large gene family of antibacterial peptides, which are critical in the host immune response against microbial invasion. The common feature of these proteins is a single WAP domain maintained by at least one four-disulfide core (4-DSC structure rich in cysteine residues. In this study, a double WAP domain (DWD-containing protein, Es-DWD1, was first cloned from the Chinese mitten crab (Eriocheirsinensis. The full-length Es-DWD1cDNA was 1193 bp, including a 411 bp open reading frame (ORF encoding 136 amino acids with a signal peptide of 22 amino acids in the N-terminus. A comparison with other reported invertebrate and vertebrate sequences revealed the presence of WAP domains characteristic of WAP superfamilies. As determined by quantitative real-time RT-PCR, Es-DWD1 transcripts were ubiquitously expressed in all tissues, but it was up-regulated in hemocytes post-challenge with pathogen-associated molecular patterns (PAMPs. The mature recombinant Es-DWD1 (rEs-DWD1 protein exhibited different binding activities to bacteria and fungus. Moreover, rEs-DWD1 could exert agglutination activities against Bacillus subtilis and Pichiapastoris and demonstrated inhibitory activities against the growth of Staphylococcus aureus, Aeromonas hydrophila and P. pastoris. Furthermore, rEs-DWD1 showed a specific protease inhibitory activity in B. subtilis. Coating of rEs-DWD1 onto agarose beads enhanced encapsulation of the beads by crab hemocytes. Collectively, the results suggest that Es-DWD1 is a double WAP domain containing protein with antimicrobial and proteinase inhibitory activities, which play significant roles in the immunity of crustaceans.

  7. Cog-Wheel Octameric Structure of RS1, the Discoidin Domain Containing Retinal Protein Associated with X-Linked Retinoschisis.

    Directory of Open Access Journals (Sweden)

    Martin Bush

    Full Text Available RS1, also known as retinoschisin, is a disulphide-linked, discoidin domain containing homo-oligomeric protein that plays a crucial role in maintaining the cellular and synaptic organization of the retina. This is highlighted by the finding that over 130 mutations in RS1 cause X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers, disruption of the photoreceptor-bipolar synapses, degeneration of photoreceptors, and severe loss in central vision. In this study, we investigated the arrangement of the RS1 subunits within the oligomer complex using single particle electron microscopy. RS1 was seen as two stacked rings with each ring displaying a symmetrical cog wheel-like structure with eight teeth or projections corresponding to the RS1 subunits. Three dimensional reconstruction and molecular modelling indicated that the discoidin domain, the principal functional unit of RS1, projects outward, and the Rs1 domain and C-terminal segment containing intermolecular disulphide bonds are present in the inner ring to form the core octameric structure. These studies provide a basis for further understanding the role of the novel core RS1 octameric complex in retinal cell biology and X-linked retinoschisis.

  8. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin (Indiana-Med)

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  9. Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

    2012-03-15

    The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

  10. Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra.

    Science.gov (United States)

    Hamaguchi-Hamada, Kayoko; Kurumata-Shigeto, Mami; Minobe, Sumiko; Fukuoka, Nozomi; Sato, Manami; Matsufuji, Miyuki; Koizumi, Osamu; Hamada, Shun

    2016-01-01

    The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH) cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR) domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

  11. Thrombospondin Type-1 Repeat Domain-Containing Proteins Are Strongly Expressed in the Head Region of Hydra.

    Directory of Open Access Journals (Sweden)

    Kayoko Hamaguchi-Hamada

    Full Text Available The head region of Hydra, the hypostome, is a key body part for developmental control and the nervous system. We herein examined genes specifically expressed in the head region of Hydra oligactis using suppression subtractive hybridization (SSH cloning. A total of 1414 subtracted clones were sequenced and found to be derived from at least 540 different genes by BLASTN analyses. Approximately 25% of the subtracted clones had sequences encoding thrombospondin type-1 repeat (TSR domains, and were derived from 17 genes. We identified 11 TSR domain-containing genes among the top 36 genes that were the most frequently detected in our SSH library. Whole-mount in situ hybridization analyses confirmed that at least 13 out of 17 TSR domain-containing genes were expressed in the hypostome of Hydra oligactis. The prominent expression of TSR domain-containing genes suggests that these genes play significant roles in the hypostome of Hydra oligactis.

  12. Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

    Science.gov (United States)

    Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

    2017-04-01

    Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

    Science.gov (United States)

    Bergmann, Christina; Brandt, Amelie; Merlevede, Benita; Hallenberger, Ludwig; Dees, Clara; Wohlfahrt, Thomas; Pötter, Sebastian; Zhang, Yun; Chen, Chih-Wei; Mallano, Tatiana; Liang, Ruifang; Kagwiria, Rosebeth; Kreuter, Alexander; Pantelaki, Ioanna; Bozec, Aline; Abraham, David; Rieker, Ralf; Ramming, Andreas; Distler, Oliver; Schett, Georg; Distler, Jörg H W

    2018-01-01

    Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFβ)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFβ. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2 . We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2 . Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No

  14. Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein

    International Nuclear Information System (INIS)

    Zhao, Hui; Wang, Hong; Liu, Huihui; Teng, Maikun; Li, Xu

    2012-01-01

    The crystallization and preliminary crystallographic studies of the carboxy-terminal domain of D. melanogaster eukaryotic translation initiation factor 5C domain-containing protein are reported. The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2B∊, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a = b = 81.05, c = 57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal

  15. Acyl CoA Binding Domain Containing 3 (ACBD3) Protein in Huntington’s Disease 
Human Skin Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, H.; Rodinová, M.; Sládková, J.; Klempíř, J.; Lišková, Irena; Motlík, Jan; Zeman, J.; Hansíková, H.; Tesařová, M.

    2015-01-01

    Roč. 78, Suppl. 2 (2015), s. 34-38 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. Liblice, 08.11.2015-10.11.2015] R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : Huntington’s disease * Acyl-CoA binding domain containing 3 protein * human skin fibroblasts Subject RIV: FH - Neurology Impact factor: 0.209, year: 2015

  16. Molluskan fasciclin-1 domain-containing protein: molecular characterizationand gene expression analysis of fasciclin 1-like protein from disk abalone (Haliotis discus discus).

    Science.gov (United States)

    Premachandra, H K A; De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Wickramaarachchi, W D N; Whang, Ilson; Lee, Jehee

    2013-06-15

    Cell-to-cell contacts play a key role in multicellular systems and organisms. Fasciclin-1 (FAS-1) is a lipid-linked membrane associated glycoprotein that is a member of a newly recognized family of cell adhesion molecules sharing features with the immunoglobulins, cadherins, integrins, and selectins. Here, we report the identification and molecular characterization of a novel FAS-1 domain-containing cDNA from disk abalone (Haliotis discus discus), including its gene expression profile and immune response to bacterial stimuli and tissue injuries. Designated as Abfac1, the 909bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33kDa and isoelectric (pI) value of 4.9. The aa sequence contains two FAS-1 domains and three conserved regions, FRa motif, H-box, and FRb motif. Phylogenetic analysis showed the closest relation to Jellyfish cell adhesion protein. In healthy abalone, Abfac1 expression is highest in hepatopancreas followed by mantle and lowest in digestive gland. In immune-stimulated abalones, relative Abfac1 mRNA expression was increased in hemocytes by ~11-fold at 48h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6h after the Listeria monocytogenes infection and by ~9-fold at 6h after the LPS injection. Similarly, tissue injuries caused significant increase of relative mRNA expression by 3.5-fold in hemocytes and by ~10-fold in mantle at 12h post-injury. These results suggest that the novel member of the FAS-1 domain-containing protein family, Abfac1, may be involved in immune response and cell adhesion in disk abalone. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  18. The periplasmic PDZ domain-containing protein Prc modulates full virulence, envelops stress responses, and directly interacts with dipeptidyl peptidase of Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Deng, Chao-Ying; Deng, Ai-Hua; Sun, Shu-Tao; Wang, Li; Wu, Jie; Wu, Yao; Chen, Xiao-Ying; Fang, Rong-Xiang; Wen, Ting-Yi; Qian, Wei

    2014-02-01

    PDZ domain-containing proteases, also known as HtrA family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell-envelope stress responses. These proteases have diverse functions through proteolysis- and nonproteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes revealed that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidence demonstrating that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell-envelope stress responses.

  19. An endoplasmic reticulum-localized Coffea arabica BURP domain-containing protein affects the response of transgenic Arabidopsis plants to diverse abiotic stresses.

    Science.gov (United States)

    Dinh, Sy Nguyen; Kang, Hunseung

    2017-11-01

    The Coffea arabica BURP domain-containing gene plays an important role in the response of transgenic Arabidopsis plants to abiotic stresses via regulating the level of diverse proteins. Although the functions of plant-specific BURP domain-containing proteins (BDP) have been determined for a few plants, their roles in the growth, development, and stress responses of most plant species, including coffee plant (Coffea arabica), are largely unknown. In this study, the function of a C. arabica BDP, designated CaBDP1, was investigated in transgenic Arabidopsis plants. The expression of CaBDP1 was highly modulated in coffee plants subjected to drought, cold, salt, or ABA. Confocal analysis of CaBDP1-GFP fusion proteins revealed that CaBDP1 is localized in the endoplasmic reticulum. The ectopic expression of CaBDP1 in Arabidopsis resulted in delayed germination of the transgenic plants under abiotic stress and in the presence of ABA. Cotyledon greening and seedling growth of the transgenic plants were inhibited in the presence of ABA due to the upregulation of ABA signaling-related genes like ABI3, ABI4, and ABI5. Proteome analysis revealed that the levels of several proteins are modulated in CaBDP1-expressing transgenic plants. The results of this study underscore the importance of BURP domain proteins in plant responses to diverse abiotic stresses.

  20. Maturation and activity of sterol regulatory element binding protein 1 is inhibited by acyl-CoA binding domain containing 3.

    Directory of Open Access Journals (Sweden)

    Yong Chen

    Full Text Available Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3, a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN. Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

  1. Crystallization and preliminary crystallographic studies of the W2 domain of Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein.

    Science.gov (United States)

    Zhao, Hui; Wang, Hong; Liu, Huihui; Teng, Maikun; Li, Xu

    2012-11-01

    The Drosophila melanogaster eukaryotic translation initiation factor 5C domain-containing protein (ECP) is composed of two independently folded domains which belong to the basic leucine-zipper and W2 domain-containing protein (BZW) family. Based on the sequence similarity between the C-terminal W2 domain of ECP and some eukaryotic translation initiation factors (such as eIF2Bℇ, eIF4γ, eIF5 etc.), ECP has been speculated to participate in the translation initiation process. Structural information on the C-terminal W2 domain of ECP would be helpful in understanding the specific cellular function of this protein. Here, the W2 domain of ECP was expressed and crystallized. Crystals grown by the hanging-drop vapour-diffusion method diffracted to 2.70 Å resolution and belonged to space group I4, with unit-cell parameters a=b=81.05, c=57.44 Å. The Matthews coefficient suggested that there was one molecule per asymmetric unit in the crystal.

  2. Evolution of double MutT/Nudix domain-containing proteins: similar domain architectures from independent gene duplication-fusion events.

    Science.gov (United States)

    Lin, Jun; Hu, Yihuai; Tian, Bing; Hua, Yuejin

    2009-10-01

    The MutT/Nudix superfamily proteins repair DNA damage and play a role in human health and disease. In this study, we examined two different cases of double MutT/Nudix domain-containing proteins from eukaryotes and prokaryotes. Firstly, these double domain proteins were discovered in Drosophila, but only single Nudix domain proteins were found in other animals. The phylogenetic tree was constructed based on the protein sequence of Nudix_N and Nudix_C from Drosophila, and Nudix from other animals. The phylogenetic analysis suggested that the double Nudix domain proteins might have undergone a gene duplication-speciation-fusion process. Secondly, two genes of the MutT family, DR0004 and DR0329, were fused by two mutT gene segments and formed double MutT domain protein genes in Deinococcus radiodurans. The evolutionary tree of bacterial MutT proteins suggested that the double MutT domain proteins in D. radiodurans probably resulted from a gene duplication-fusion event after speciation. Gene duplication-fusion is a basic and important gene innovation mechanism for the evolution of double MutT/Nudix domain proteins. Independent gene duplication-fusion events resulted in similar domain architectures of different double MutT/Nudix domain proteins.

  3. Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2.

    Science.gov (United States)

    Wu, Di; Zhu, Xiaoxi; Jimenez-Cowell, Kevin; Mold, Alexander J; Sollecito, Christopher C; Lombana, Nicholas; Jiao, Meng; Wei, Qize

    2015-04-10

    Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins.

    Science.gov (United States)

    Cirl, Christine; Wieser, Andreas; Yadav, Manisha; Duerr, Susanne; Schubert, Sören; Fischer, Hans; Stappert, Dominik; Wantia, Nina; Rodriguez, Nuria; Wagner, Hermann; Svanborg, Catharina; Miethke, Thomas

    2008-04-01

    Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.

  5. Anchoring skeletal muscle development and disease: The role of ankyrin repeat domain containing proteins in muscle physiology

    NARCIS (Netherlands)

    J-M. Tee (Jin-Ming); M.P. Peppelenbosch (Maikel)

    2010-01-01

    textabstractThe ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary

  6. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, H Y; Cai, M

    1996-09-01

    Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

  7. The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Goldová, Jana; Petříček, Miroslav; Benada, Oldřich; Kofroňová, Olga; Rampírová, Petra; Petříčková, Kateřina; Branny, Pavel

    2013-01-01

    Roč. 9, č. 6 (2013), s. 1453-1469 ISSN 1742-206X R&D Projects: GA MŠk LH12191; GA MŠk LH12055; GA MŠk 2B08064 Institutional support: RVO:61388971 Keywords : AERIAL MYCELIUM FORMATION * WD-REPEAT PROTEINS * BIOSYNTHESIS GENE-CLUSTER Subject RIV: EE - Microbiology, Virology Impact factor: 3.183, year: 2013

  8. Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins

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    Katherine S. Bridge

    2017-07-01

    Full Text Available As core components of the microRNA-induced silencing complex (miRISC, Argonaute (AGO proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390 on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

  9. Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development.

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    Chung-Hsiang Yang

    Full Text Available BACKGROUND: The zona pellucida (ZP domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear. METHODOLOGY/PRINCIPAL FINDINGS: We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised. CONCLUSIONS/SIGNIFICANCE: Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.

  10. Zona pellucida domain-containing protein β-tectorin is crucial for zebrafish proper inner ear development.

    Science.gov (United States)

    Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Chen, Gen-Der; Liao, Wei-Hao; Chen, Yi-Chung; Huang, Kai-Yun; Hwang, Pung-Pung; Hwang, Sheng-Ping L; Huang, Chang-Jen

    2011-01-01

    The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear. We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised. Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function.

  11. The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity.

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    Uchida, Kai; Akashi, Tomoyoshi; Aoki, Toshio

    2017-02-01

    Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this step, 2'-hydroxyisoflavanol 4,2'-dehydratase or pterocarpan synthase (PTS), remains as an unidentified 'missing link'. This last ring formation is assumed to be the key step in determining the stereochemistry of pterocarpans, which plays a role in their antimicrobial activity. In this study, a cDNA clone encoding PTS from Glycyrrhiza echinata (GePTS1) was identified through functional expression fractionation screening of a cDNA library, which requires no sequence information, and orthologs from soybean (GmPTS1) and Lotus japonicus (LjPTS1) were also identified. These proteins were heterologously expressed in Escherichia coli and biochemically characterized. Surprisingly, the proteins were found to include amino acid motifs characteristic of dirigent proteins, some of which control stereospecific phenoxy radical coupling in lignan biosynthesis. The stereospecificity of substrates and products was examined using four substrate stereoisomers with hydroxy and methoxy derivatives at C-4'. The results showed that the 4R configuration was essential for the PTS reaction, and (-)- and (+)-pterocarpans were produced depending on the stereochemistry at C-3. In suspension-cultured soybean cells, levels of the GmPTS1 transcript increased temporarily prior to the peak in phytoalexin accumulation, strongly supporting the possible involvement of PTS in pterocarpan biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.

  12. The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules.

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    Sung, Michael; Poon, Gregory M K; Gariépy, Jean

    2006-03-01

    Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.

  13. Regulation of plant defense responses in Arabidopsis by EDR2, a PH and START domain-containing protein.

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    Tang, Dingzhong; Ade, Jules; Frye, Catherine A; Innes, Roger W

    2005-10-01

    We have identified an Arabidopsis mutant that displays enhanced disease resistance (edr2) to the biotrophic powdery mildew pathogen Erysiphe cichoracearum. Inhibition of fungal growth on edr2 mutant leaves occurred at a late stage of the infection process and coincided with formation of necrotic lesions approximately 5 days after inoculation. Double-mutant analysis revealed that edr2-mediated resistance is suppressed by mutations that inhibit salicylic acid (SA)-induced defense signaling, including npr1, pad4 and sid2, demonstrating that edr2-mediated disease resistance is dependent on SA. However, edr2 showed normal responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. EDR2 appears to be constitutively transcribed in all tissues and organs and encodes a novel protein, consisting of a putative pleckstrin homology (PH) domain and a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and contains an N-terminal mitochondrial targeting sequence. The PH and START domains are implicated in lipid binding, suggesting that EDR2 may provide a link between lipid signaling and activation of programmed cell death mediated by mitochondria.

  14. Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein

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    Patra, Mahesh Chandra; Choi, Sangdun

    2018-01-01

    The toll/interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) plays an important role in the toll-like receptor (TLR) 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) on the plasma membrane and PI (3,4,5)-trisphosphate (PIP3) on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP’s membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein–protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD) for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM) contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32), additional positively charged residues (K34, K35, and R36) showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP’s affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond) and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP’s membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases. PMID:29434596

  15. Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein

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    Mahesh Chandra Patra

    2018-01-01

    Full Text Available The toll/interleukin 1 receptor (TIR domain-containing adaptor protein (TIRAP plays an important role in the toll-like receptor (TLR 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI 4,5-bisphosphate (PIP2 on the plasma membrane and PI (3,4,5-trisphosphate (PIP3 on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP’s membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein–protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32, additional positively charged residues (K34, K35, and R36 showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP’s affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP’s membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.

  16. Regulation and Essentiality of the StAR-related Lipid Transfer (START) Domain-containing Phospholipid Transfer Protein PFA0210c in Malaria Parasites.

    Science.gov (United States)

    Hill, Ross J; Ringel, Alessa; Knuepfer, Ellen; Moon, Robert W; Blackman, Michael J; van Ooij, Christiaan

    2016-11-11

    StAR-related lipid transfer (START) domains are phospholipid- or sterol-binding modules that are present in many proteins. START domain-containing proteins (START proteins) play important functions in eukaryotic cells, including the redistribution of phospholipids to subcellular compartments and delivering sterols to the mitochondrion for steroid synthesis. How the activity of the START domain is regulated remains unknown for most of these proteins. The Plasmodium falciparum START protein PFA0210c (PF3D7_0104200) is a broad-spectrum phospholipid transfer protein that is conserved in all sequenced Plasmodium species and is most closely related to the mammalian START proteins STARD2 and STARD7. PFA0210c is unusual in that it contains a signal sequence and a PEXEL export motif that together mediate transfer of the protein from the parasite to the host erythrocyte. The protein also contains a C-terminal extension, which is very uncommon among mammalian START proteins. Whereas the biochemical properties of PFA0210c have been characterized, the function of the protein remains unknown. Here, we provide evidence that the unusual C-terminal extension negatively regulates phospholipid transfer activity. Furthermore, we use the genetically tractable Plasmodium knowlesi model and recently developed genetic technology in P. falciparum to show that the protein is essential for growth of the parasite during the clinically relevant asexual blood stage life cycle. Finally, we show that the regulation of phospholipid transfer by PFA0210c is required in vivo, and we identify a potential second regulatory domain. These findings provide insight into a novel mechanism of regulation of phospholipid transfer in vivo and may have important implications for the interaction of the malaria parasite with its host cell. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The effect of the disulfideisomerase domain containing protein in the defense against polyhexamethylene biguanide of highly tolerant Acanthamoeba at the trophozoite stage

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    Fu-Chin Huang

    2016-12-01

    Full Text Available Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing a blinding keratitis and fatal granulomatous encephalitis. Current treatment generally involves an hourly application of polyhexamethylene biguanide (PHMB over a period of several days but this is not entirely effective against all strains/isolates. The tolerance mechanisms of PHMB in Acanthamoeba cells remain unclear. In this study, we found that the mRNA expression level of disulfideisomerase domain containing protein (PDI increased rapidly in surviving cells of the highly PHMB-tolerant Acanthamoeba castellanii strain, NCKH_D, during PHMB treatment, but not in the ATCC standard strain. After PDI-specific silencing, NCKH_D was found to be more vulnerable to PHMB treatment. The results described above show that PDI is an important gene for PHMB tolerance ability in a highly PHMB-tolerant strain of Acanthamoeba and provide a new insight for more efficient medicine development for Acanthamoeba keratitis.

  18. Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri

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    Xu Qianghua

    2012-12-01

    Full Text Available Abstract Background Zona pellucida domain-containing proteins (ZP proteins have been identified as the principle constituents of the egg coat (EC of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata. Results Five ZP proteins (BbZP1-5 were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates

  19. Characterization of big bang, a novel gene encoding for PDZ domain-containing proteins that are dynamically expressed throughout Drosophila development.

    Science.gov (United States)

    Kim, Sabrina Y; Renihan, Maia K; Boulianne, Gabrielle L

    2006-06-01

    PDZ (PSD-95, Discs-large, ZO-1) domain proteins often function as scaffolding proteins and have been shown to play important roles in diverse cellular processes such as the establishment and maintenance of cell polarity, and signal transduction. Here, we report the identification and cloning of a novel Drosophila melanogaster gene that is predicted to produce several different PDZ domain-containing proteins through alternative promoter usage and alternative splicing. This gene, that we have named big bang (bbg), was first identified as C96-GAL4, a GAL4 enhancer trap line that was generated in our lab. To further characterize bbg, its expression pattern was examined in ovaries, embryos, and late third instar larvae using UAS reporter gene constructs, in situ hybridization, or immunocytochemistry. In addition, the expression of alternatively spliced transcripts was examined in more detail using in situ hybridization. We find that during embryogenesis bbg is predominantly expressed in the developing gut, but it is also expressed in external sensory organs found in the epidermis. In the late third instar larva, bbg is expressed along the presumptive wing margin in the wing disc, broadly in the eye disc, and in other imaginal discs as well as in the brain. The expression patterns observed are dynamic and specific during development, suggesting that like other genes that encode for several different PDZ domain protein isoforms, bbg likely plays important roles in multiple developmental processes.

  20. Identification of DEP domain-containing proteins by a machine learning method and experimental analysis of their expression in human HCC tissues

    Science.gov (United States)

    Liao, Zhijun; Wang, Xinrui; Zeng, Yeting; Zou, Quan

    2016-12-01

    The Dishevelled/EGL-10/Pleckstrin (DEP) domain-containing (DEPDC) proteins have seven members. However, whether this superfamily can be distinguished from other proteins based only on the amino acid sequences, remains unknown. Here, we describe a computational method to segregate DEPDCs and non-DEPDCs. First, we examined the Pfam numbers of the known DEPDCs and used the longest sequences for each Pfam to construct a phylogenetic tree. Subsequently, we extracted 188-dimensional (188D) and 20D features of DEPDCs and non-DEPDCs and classified them with random forest classifier. We also mined the motifs of human DEPDCs to find the related domains. Finally, we designed experimental verification methods of human DEPDC expression at the mRNA level in hepatocellular carcinoma (HCC) and adjacent normal tissues. The phylogenetic analysis showed that the DEPDCs superfamily can be divided into three clusters. Moreover, the 188D and 20D features can both be used to effectively distinguish the two protein types. Motif analysis revealed that the DEP and RhoGAP domain was common in human DEPDCs, human HCC and the adjacent tissues that widely expressed DEPDCs. However, their regulation was not identical. In conclusion, we successfully constructed a binary classifier for DEPDCs and experimentally verified their expression in human HCC tissues.

  1. Major facilitator superfamily domain-containing protein 2a (MFSD2A has roles in body growth, motor function, and lipid metabolism.

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    Justin H Berger

    Full Text Available The metabolic adaptations to fasting in the liver are largely controlled by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARα, where PPARα upregulates genes encoding the biochemical pathway for β-oxidation of fatty acids and ketogenesis. As part of an effort to identify and characterize nutritionally regulated genes that play physiological roles in the adaptation to fasting, we identified Major facilitator superfamily domain-containing protein 2a (Mfsd2a as a fasting-induced gene regulated by both PPARα and glucagon signaling in the liver. MFSD2A is a cell-surface protein homologous to bacterial sodium-melibiose transporters. Hepatic expression and turnover of MFSD2A is acutely regulated by fasting/refeeding, but expression in the brain is constitutive. Relative to wildtype mice, gene-targeted Mfsd2a knockout mice are smaller, leaner, and have decreased serum, liver and brown adipose triglycerides. Mfsd2a knockout mice have normal liver lipid metabolism but increased whole body energy expenditure, likely due to increased β-oxidation in brown adipose tissue and significantly increased voluntary movement, but surprisingly exhibited a form of ataxia. Together, these results indicate that MFSD2A is a nutritionally regulated gene that plays myriad roles in body growth and development, motor function, and lipid metabolism. Moreover, these data suggest that the ligand(s that are transported by MFSD2A play important roles in these physiological processes and await future identification.

  2. SET domain-containing protein 5 is required for expression of primordial germ cell specification-associated genes in murine embryonic stem cells.

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    Yu, Seung Eun; Kim, Min Seong; Park, Su Hyung; Yoo, Byong Chul; Kim, Kyung Hee; Jang, Yeun Kyu

    2017-07-01

    Primordial germ cell (PGC) specification is one of the most fundamental processes in developmental biology. Because PGCs are a common source of both gametes, generation of PGCs from embryonic stem cells (ESCs) is a useful model for analysing the germ line lineage. Although several studies focused on the role of epigenetic regulation on PGC differentiation from ESCs in vitro have been published, germ line commitment remains poorly understood. Here, we show that SET domain-containing protein (Setd5), which has a previously unknown function, is essential for regulating germ cell-associated genes in murine ESCs (mESCs). Even though Setd5 knockdown with 3 distinct shRNAs did not affect expression of pluripotency genes or levels of global histone methylation, all 3 shRNAs significantly diminished the expression of early and late-stage PGC-associated genes. Furthermore, our immunoprecipitation assay showed that Setd5 can interact with Tbl1xr1 and Ctr9, which are components of 2 different transcriptional regulatory complexes, namely, NcoR1 corepressor complex and Paf1 complex, respectively, in mESCs. Taken together, our data suggest that Setd5 is required for maintaining PGC-associated genes and Setd5-associated protein complexes containing Tbl1xr1 and Ctr9, which in turn are likely involved in regulating germ cell-related genes in mESCs. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5'-UTR.

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    McAninch, Damian S; Heinaman, Ashley M; Lang, Cara N; Moss, Kathryn R; Bassell, Gary J; Rita Mihailescu, Mihaela; Evans, Timothy L

    2017-07-25

    G quadruplex structures have been predicted by bioinformatics to form in the 5'- and 3'-untranslated regions (UTRs) of several thousand mature mRNAs and are believed to play a role in translation regulation. Elucidation of these roles has primarily been focused on the 3'-UTR, with limited focus on characterizing the G quadruplex structures and functions in the 5'-UTR. Investigation of the affinity and specificity of RNA binding proteins for 5'-UTR G quadruplexes and the resulting regulatory effects have also been limited. Among the mRNAs predicted to form a G quadruplex structure within the 5'-UTR is the survival motor neuron domain containing 1 (SMNDC1) mRNA, encoding a protein that is critical to the spliceosome. Additionally, this mRNA has been identified as a potential target of the fragile X mental retardation protein (FMRP), whose loss of expression leads to fragile X syndrome. FMRP is an RNA binding protein involved in translation regulation that has been shown to bind mRNA targets that form G quadruplex structures. In this study we have used biophysical methods to investigate G quadruplex formation in the 5'-UTR of SMNDC1 mRNA and analyzed its interactions with FMRP. Our results show that SMNDC1 mRNA 5'-UTR forms an intramolecular, parallel G quadruplex structure comprised of three G quartet planes, which is bound specifically by FMRP both in vitro and in mouse brain lysates. These findings suggest a model by which FMRP might regulate the translation of a subset of its mRNA targets by recognizing the G quadruplex structure present in their 5'-UTR, and affecting their accessibility by the protein synthesis machinery.

  4. A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

    International Nuclear Information System (INIS)

    Rudchenko, Sergei; Trakht, Ilya; Scanlan, Matthew; Kalantarov, Gavreel; Yavelsky, Victoria; Levy, Chen; Estabrook, Alison; Old, Lloyd; Chan, Gerald L; Lobel, Leslie

    2008-01-01

    We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification. By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein. We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification

  5. Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens.

    Science.gov (United States)

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Jayasinghe, J D H E; Godahewa, G I; Park, Hae-Chul; Lee, Jehee

    2016-09-01

    The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qDC3 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor-joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different

  6. Can signal peptide-CUB-EGF domain-containing protein (SCUBE) levels be a marker of angiogenesis in patients with psoriasis?

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    Capkin, Arzu Aydın; Demir, Selim; Mentese, Ahmet; Bulut, Çağlar; Ayar, Ahmet

    2017-04-01

    Angiogenesis is an important process being involved in the pathogenesis of psoriasis and promises new potential parameter for diagnosis and screening of treatment. This study investigated the levels of signal peptide-CUB-EGF (epidermal growth factor-like protein) family domain-containing protein (SCUBE) 1 and 3. Potential value as a novel marker of angiogenesis in patients with psoriasis is also evaluated by assessing possible relation of SCUBE-1 and 3 with disease activity in conjunction with vascular endothelial growth factor (VEGF) levels, as an established marker of angiogenesis. Forty-eight patients with psoriasis (aged >18 years) and 48 age- and gender-matched healthy controls were included. Detailed information was obtained through history and physical examination. Psoriasis area and severity index (PASI) scores were calculated. Blood SCUBE 1 and 3, and VEGF levels were measured by enzyme-linked immunosorbent assay. The mean PASI score of the patients was 6.7 ± 4.1. Patients' serum SCUBE 1 and 3 and VEGF levels were significantly higher than those of the controls (P = 0.001). The sensitivity and specificity were calculated as 83 and 62% for the 0.67 ng/ml cut-off level of SCUBE 1, and 63 and 71% for the 2.57 ng/ml cut-off level of SCUBE 3, respectively. A cut-off VEGF level of 310 ng/mL predicted the presence of psoriasis with a sensitivity of 50% and specificity of 77%. The results of this pioneering study indicate that SCUBE protein family appears to have a probable role in the pathogenesis and angiogenesis development in psoriasis and SCUBE 1 and 3 may be novel markers of angiogenesis in psoriasis.

  7. Interleukin-4- and NACHT, LRR and PYD domains-containing protein 3-independent mechanisms of alum enhanced T helper type 2 responses on basophils.

    Science.gov (United States)

    Huang, Feng-Juan; Ma, Yi-Lei; Tang, Ruo-Yu; Gong, Wen-Ci; Li, Jun; Chen, Chun-Xia; Yin, Lan; Chen, Xiao-Ping

    2016-10-01

    Aluminium hydroxide (alum), the most widely used adjuvant in human and animal vaccines, has long been known to promote T helper type 2 (Th2) responses and Th2-associated humoral responses, but the mechanisms have remained poorly understood. In this study, we explored whether alum is able to directly modulate antigen-presenting cells to enhance their potency for Th2 polarization. We found that alum treatment of dendritic cells failed to show any Th2-promoting activities. In contrast, alum was able to enhance the capacity of basophils to induce Th2 cells. When basophils from interleukin-4 (IL-4) knockout mice were examined, the intrinsic Th2-promoting activities by basophils were largely abrogated, but the alum-enhanced Th2-promoting activities on basophils were still detectable. More importantly, Th2-promoting adjuvant activities by alum found in IL-4 knockout mice were also largely reduced when basophils were depleted by antibody administration. Therefore, basophils can mediate Th2-promoting activities by alum both in vitro and in vivo through IL-4-independent mechanisms. Further studies revealed that secreted soluble molecules from alum-treated basophils were able to confer the Th2-promoting activities, and neutralization of thymic stromal lymphopoietin or IL-25 attenuated the IL-4-independent development of Th2 cells elicited by alum-treated basophils. Finally, alum was able to activate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in murine basophils in the same way as alum in professional antigen-presenting cells, but NLRP3 was not required for Th2-promoting activities on basophils by alum in vitro. These results demonstrated that alum can enhance the capacities of basophils to polarize Th2 cells via IL-4- and NLRP3-independent pathways. © 2016 John Wiley & Sons Ltd.

  8. A cupin domain-containing protein with a quercetinase activity (VdQase regulates Verticillium dahliae’s pathogenicity and contributes to counteracting host defenses

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    Abdel eElHadrami

    2015-06-01

    Full Text Available We previously identified rutin as part of potato root responses to its pathogen Verticillium dahliae. Rutin was directly toxic to the pathogen at doses greater than 160 μM, a threshold below which many V. dahliae pathogenicity-related genes were up-regulated. We identified and characterized a cupin domain-containing protein (VdQase with a dioxygenase activity and a potential role in V. dahliae-potato interactions. The pathogenicity of VdQase knock-out mutants generated through Agrobacterium tumefasciens-mediated transformation was significantly reduced on susceptible potato cultivar Kennebec compared to wild type isolates. Fluorescence microscopy revealed a higher accumulation of flavonols in the stems of infected potatoes and a higher concentration of rutin in the leaves in response to the VdQase mutants as compared to wild type isolates. This, along with the HPLC characterization of high residual and non-utilized quercetin in presence of the knockout mutants, indicates the involvement of VdQase in the catabolism of quercetin and possibly other flavonols in planta. Quantification of Salicylic and Jasmonic Acids (SA, JA in response to the mutants versus wild type isolates revealed involvement of VdQase in the interference with signaling, suggesting a role in pathogenicity. It is hypothesized that the by-product of dioxygenation 2-protocatechuoylphloroglucinolcarboxylic acid, after dissociating into phloroglucinol and protocatechuoyl moieties, becomes a starting point for benzoic acid and SA, thereby interfering with the JA pathway and affecting the interaction outcome. These events may be key factors for V. dahliae in countering potato defenses and becoming notorious in the rhizosphere.

  9. The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

    OpenAIRE

    Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

    2015-01-01

    Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

  10. The RST and PARP-like domain containing SRO protein family: analysis of protein structure, function and conservation in land plants

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    Salojärvi Jarkko

    2010-03-01

    Full Text Available Abstract Background The SROs (SIMILAR TO RCD-ONE are a group of plant-specific proteins which have important functions in stress adaptation and development. They contain the catalytic core of the poly(ADP-ribose polymerase (PARP domain and a C-terminal RST (RCD-SRO-TAF4 domain. In addition to these domains, several, but not all, SROs contain an N-terminal WWE domain. Results SROs are present in all analyzed land plants and sequence analysis differentiates between two structurally distinct groups; cryptogams and monocots possess only group I SROs whereas eudicots also contain group II. Group I SROs possess an N-terminal WWE domain (PS50918 but the WWE domain is lacking in group II SROs. Group I domain structure is widely represented in organisms as distant as humans (for example, HsPARP11. We propose a unified nomenclature for the SRO family. The SROs are able to interact with transcription factors through the C-terminal RST domain but themselves are generally not regulated at the transcriptional level. The most conserved feature of the SROs is the catalytic core of the poly(ADP-ribose polymerase (PS51059 domain. However, bioinformatic analysis of the SRO PARP domain fold-structure and biochemical assays of AtRCD1 suggested that SROs do not possess ADP-ribosyl transferase activity. Conclusions The SROs are a highly conserved family of plant specific proteins. Sequence analysis of the RST domain implicates a highly preserved protein structure in that region. This might have implications for functional conservation. We suggest that, despite the presence of the catalytic core of the PARP domain, the SROs do not possess ADP-ribosyl transferase activity. Nevertheless, the function of SROs is critical for plants and might be related to transcription factor regulation and complex formation.

  11. Targeted interfering DEP domain containing 1 protein induces apoptosis in A549 lung adenocarcinoma cells through the NF-κB signaling pathway.

    Science.gov (United States)

    Wang, Qingqing; Li, Aili; Jin, Junfei; Huang, Guojin

    2017-01-01

    Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611-628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611-628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611-628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611-628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611-628 peptide is a potent apoptosis inducer in A549 cells.

  12. The C-type lectin-like domain containing proteins Clec-39 and Clec-49 are crucial for Caenorhabditis elegans immunity against Serratia marcescens infection.

    Science.gov (United States)

    Miltsch, S M; Seeberger, P H; Lepenies, B

    2014-07-01

    Caenorhabditis elegans exhibits protective immunity against a variety of fungal and bacterial pathogens. Since C. elegans lacks an adaptive immune system, pathogen recognition is mediated entirely by innate immunity. To date, little is known about the involvement of pattern recognition receptors (PRRs) in pathogen sensing as part of the C. elegans immunity. C-type lectin-like domain (CTLD) containing proteins represent a superfamily of PRRs. A large number of genes encoding for CTLD proteins are present in the C. elegans genome, however the role of CTLD proteins in bacterial recognition and antibacterial immunity has not yet been determined. In this study, we investigated the function of selected C. elegans CTLD proteins during infection with the Gram-negative bacterium Serratia marcescens. Wild-type and CTLD gene-deficient C. elegans strains were compared in their susceptibility to S. marcescens infection. Interestingly, survival and egg laying were significantly reduced in strains deficient for clec-39 and clec-49 indicating a role for both CTLD proteins in C. elegans immune defense against bacteria as evidenced by using S. marcescens infection. Binding studies with recombinantly expressed Clec-39-Fc and Clec-49-Fc fusion proteins revealed that both CTLD proteins recognized live bacteria in a Ca(2+)-independent manner. This study provides insight into the role of CTLD proteins in C. elegans immunity and demonstrates their function during bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. The non-canonical Wnt/PKC pathway regulates mitochondrial dynamics through degradation of the arm-like domain-containing protein Alex3.

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    Román Serrat

    Full Text Available The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3 that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation.

  14. The Non-Canonical Wnt/PKC Pathway Regulates Mitochondrial Dynamics through Degradation of the Arm-Like Domain-Containing Protein Alex3

    Science.gov (United States)

    Mirra, Serena; Quevedo, Martí; Garcia-Fernàndez, Jordi; Ulloa, Fausto; Burgaya, Ferrán; Soriano, Eduardo

    2013-01-01

    The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation. PMID:23844091

  15. The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions.

    Science.gov (United States)

    Beaury, Michael; Velagapudi, Uday Kiran; Weber, Sarah; Soto, Cassandra; Talele, Tanaji T; Nichols, Daniel Brian

    2017-08-01

    The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNβ activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1 = R67A D69A; DED2 = R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

  16. BcCFEM1, a CFEM Domain-Containing Protein with Putative GPI-Anchored Site, Is Involved in Pathogenicity, Conidial Production, and Stress Tolerance in Botrytis cinerea

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    Wenjun Zhu

    2017-09-01

    Full Text Available We experimentally isolated and characterized a CFEM protein with putative GPI-anchored site BcCFEM1 in Botrytis cinerea. BcCFEM1 contains a CFEM (common in several fungal extracellular membrane proteins domain with the characteristic eight cysteine residues at N terminus, and a predicted GPI modification site at C terminus. BcCFEM1 was significantly up-regulated during early stage of infection on bean leaves and induced chlorosis in Nicotiana benthamiana leaves using Agrobacterium infiltration method. Targeted deletion of BcCFEM1 in B. cinerea affected virulence, conidial production and stress tolerance, but not growth rate, conidial germination, colony morphology, and sclerotial formation. However, over expression of BcCFEM1 did not make any observable phenotype change. Therefore, our data suggested that BcCFEM1 contributes to virulence, conidial production, and stress tolerance. These findings further enhance our understanding on the sophisticated pathogenicity of B. cinerea beyond necrotrophic stage, highlighting the importance of CFEM protein to B. cinerea and other broad-host-range necrotrophic pathogens.

  17. A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation.

    Science.gov (United States)

    Sinharoy, Senjuti; Liu, Chengwu; Breakspear, Andrew; Guan, Dian; Shailes, Sarah; Nakashima, Jin; Zhang, Shulan; Wen, Jiangqi; Torres-Jerez, Ivone; Oldroyd, Giles; Murray, Jeremy D; Udvardi, Michael K

    2016-04-01

    The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca(2+) responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall. © 2016 American Society of Plant Biologists. All Rights Reserved.

  18. Redistribution of demethylated RNA helicase A during foot-and-mouth disease virus infection: Role of Jumonji C-domain containing protein 6 in RHA demethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Conderino, Joseph S.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2014-03-15

    Previously, RNA helicase A (RHA) re-localization from the nucleus to the cytoplasm in foot-and-mouth disease virus (FMDV) infected cells was shown to coincide with loss of RHA methylated arginine residues at its C-terminus. The potential interaction between RHA and Jumonji C-domain (JmjC) protein 6 (JMJD6) arginine demethylase in infected cells was investigated. Treatment with N-oxalylglycine (NOG) inhibitor of JmjC demethylases prevented FMDV-induced RHA demethylation and re-localization, and also decreased viral protein synthesis and virus titers. Physical interaction between JMJD6 and RHA was demonstrated via reciprocal co-immunoprecipitation, where RHA preferentially bound JMJD6 monomers. Nuclear efflux of demethylated RHA (DM-RHA) coincided with nuclear influx of JMJD6, which was not observed using another picornavirus. A modified biochemical assay demonstrated JMJD6 induced dose-dependent demethylation of RHA and two RHA-derived isoforms, which could be inhibited by NOG. We propose a role for JMJD6 in RHA demethylation stimulated by FMDV, that appears to facilitate virus replication. - Highlights: • We examined the role of JMJD6 in FMDV-induced RHA demethylation process. • Using an arginine demethylation assay showed that JMJD6 is involved in RHA demethylation. • A demethylases inhibitor reduced cytoplasmic accumulation of RHA and FMDV titers.

  19. The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane.

    Science.gov (United States)

    Nguyen, Hong Hanh; Lee, Myoung Hui; Song, Kyungyoung; Ahn, Gyeongik; Lee, Jihyeong; Hwang, Inhwan

    2018-01-06

    Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

  20. Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

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    In Sil Jeong

    Full Text Available Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD PHOSPHATASE-LIKE 1 (CPL1 regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds RNA binding motifs (dsRBMs at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3 as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

  1. H2O2 production rate in Lactobacillus johnsonii is modulated via the interplay of a heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain containing protein.

    Directory of Open Access Journals (Sweden)

    Ricardo B Valladares

    2015-07-01

    Full Text Available Host and commensals crosstalk, mediated by reactive oxygen species (ROS, has triggered a growing scientific interest to understand the mechanisms governing such interaction. However, the majority of the scientific studies published do not evaluate the ROS production by commensals bacteria. In this context we recently showed that Lactobacillus johnsonii N6.2, a strain of probiotic value, modulates the activity of the critical enzymes 2,3-indoleamine dioxygenase via H2O2 production. L. johnsonii N6.2 by decreasing IDO activity, is able to modify the tryptophan/kynurenine ratio in the host blood with further systemic consequences. Understanding the mechanisms of H2O2 production is critical to predict the probiotic value of these strains and to optimize bacterial biomass production in industrial processes. We performed a transcriptome analysis to identify genes differentially expressed in L. johnsonii N6.2 cells collected from cultures grown under different aeration conditions. Herein we described the biochemical characteristics of a heterodimeric FMN reductase (FRedA/B whose in vitro activity is controlled by LjPAS protein with a typical Per-Arnst-Sim (PAS sensor domain. Interestingly, LjPAS is fused to the FMN reductase domains in other lactobacillaceae. In L. johnsonii, LjPAS is encoded by an independent gene which expression is repressed under anaerobic conditions (>3 fold. Purified LjPAS was able to slow down the FRedA/B initial activity rate when the holoenzyme precursors (FredA, FredB and FMN were mixed in vitro. Altogether the results obtained suggest that LjPAS module regulates the H2O2 production helping the cells to minimize oxidative stress in response to environmental conditions.

  2. Hi-C First Results

    Science.gov (United States)

    Cirtain, Jonathan

    2013-01-01

    Hi-C obtained the highest spatial and temporal resolution observatoins ever taken in the solar corona. Hi-C reveals dynamics and structure at the limit of its temporal and spatial resolution. Hi-C observed ubiquitous fine-scale flows consistent with the local sound speed.

  3. Human T-Cell Leukemia Virus Type I-Mediated Repression of PDZ-LIM Domain-Containing Protein 2 Involves DNA Methylation But Independent of the Viral Oncoprotein Tax

    Directory of Open Access Journals (Sweden)

    Pengrong Yan

    2009-10-01

    Full Text Available Human T-cell leukemia virus type I (HTLV-I is the etiological agent of adult T-cell leukemia (ATL. Our recent studies have shown that one important mechanism of HTLV-I-Mediated tumorigenesis is through PDZ-LIM domain-containing protein 2 (PDLIM2 repression, although the involved mechanism remains unknown. Here, we further report that HTLV-I-Mediated PDLIM2 repression was a pathophysiological event and the PDLIM2 repression involved DNA methylation. Whereas DNA methyltransferases 1 and 3b but not 3a were upregulated in HTLV-I-transformed T cells, the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC restored PDLIM2 expression and induced death of these malignant cells. Notably, the PDLIM2 repression was independent of the viral regulatory protein Tax because neither short-term induction nor long-term stable expression of Tax could downregulate PDLIM2 expression. These studies provide important insights into PDLIM2 regulation, HTLV-I leukemogenicity, long latency, and cancer health disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, these studies also suggest potential therapeutic strategies for ATL.

  4. RHOMBOID DOMAIN CONTAINING 2 (RHBDD2)

    Science.gov (United States)

    Abba, MC; Lacunza, E; Nunez, MI; Colussi, A; Isla-Larrain, M; Segal-Eiras, A; Croce, MV; Aldaz, CM

    2009-01-01

    In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p= 0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression. PMID:19616622

  5. The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

    International Nuclear Information System (INIS)

    Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

    2015-01-01

    The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients

  6. The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients.

    Science.gov (United States)

    Englund, Emelie; Reitsma, Bart; King, Ben C; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G; Jirström, Karin; Blom, Anna M

    2015-10-19

    The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

  7. Cross-talk between the glucocorticoid receptor and MyoD family inhibitor domain-containing protein provides a new mechanism for generating tissue-specific responses to glucocorticoids.

    Science.gov (United States)

    Oakley, Robert H; Busillo, John M; Cidlowski, John A

    2017-04-07

    Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic. The molecular factors that govern tissue specificity of glucocorticoids, however, are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast two-hybrid assay. The MyoD family inhibitor domain-containing protein (MDFIC) was identified as a binding partner for GR. MDFIC associated with GR in the cytoplasm of cells, and treatment with glucocorticoids resulted in the dissociation of the GR-MDFIC complex. To investigate the function of the GR-MDFIC interaction, we performed a genome-wide microarray in intact and MDFIC-deficient A549 cells that were treated with glucocorticoids. A large cohort of genes was differentially regulated by GR depending on the presence or absence of MDFIC. These gene changes were strongly associated with inflammation, and glucocorticoid regulation of the inflammatory response was altered in MDFIC-deficient cells. At a molecular level, the interaction of MDFIC with GR altered the phosphorylation status of the receptor. We demonstrate in COS-1 cells that changes in receptor phosphorylation underlie the ability of MDFIC to regulate the transcriptional activity of GR. Finally, we show that GR directly represses the MDFIC gene, revealing a negative feedback loop by which glucocorticoids limit MDFIC activity. These findings identify a new binding partner for cytoplasmic GR that modulates the receptor transcriptome and contributes to the tissue-specific actions of glucocorticoids. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-α production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1.

    Science.gov (United States)

    Pedroza, Luis A; Kumar, Vipul; Sanborn, Keri B; Mace, Emily M; Niinikoski, Harri; Nadeau, Kari; Vasconcelos, Dewton de Moraes; Perez, Elena; Jyonouchi, Soma; Jyonouchi, Harumi; Banerjee, Pinaki P; Ruuskanen, Olli; Condino-Neto, Antonio; Orange, Jordan S

    2012-02-01

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  9. Se14, encoding a JmjC domain-containing protein, plays key roles in long-day suppression of rice flowering through the demethylation of H3K4me3 of RFT1.

    Directory of Open Access Journals (Sweden)

    Takayuki Yokoo

    Full Text Available Floral transition from the vegetative to the reproductive growth phase is a major change in the plant life cycle and a key factor in reproductive success. In rice (Oryza sativa L., a facultative short-day plant, numerous flowering time and flower formation genes that control floral transition have been identified and their physiological effects and biochemical functions have been clarified. In the present study, we used a Se14-deficient mutant line (HS112 and other flowering mutant lines to investigate the photoperiodic response, chromosomal location and function in the photoperiod sensitivity of the Se14 gene. We also studied the interactive effects of this locus with other crucial flowering time genes. We found that Se14 is independent of the known photoperiod-sensitive genes, such as Hd1 and Ghd7, and is identical to Os03g0151300, which encodes a Jumonji C (JmjC domain-containing protein. Expression analysis revealed that the expressions of RFT1, a floral initiator known as a "florigen-like gene", and Ehd1 were up-regulated in HS112, whereas this up-regulation was not observed in the original variety of 'Gimbozu'. ChIP assays of the methylation states of histone H3 at lysine 4 (H3K4 revealed that the trimethylated H3K4 in the promoter region of the RFT1 chromatin was significantly increased in HS112. We conclude that Se14 is a novel photoperiod-sensitivity gene that has a suppressive effect on floral transition (flowering time under long day-length conditions through the modification of chromatin structure by H3K4me3 demethylation in the promoter region of RFT1.

  10. Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice

    DEFF Research Database (Denmark)

    Costinean, Stefan; Sandhu, Sukhinder K; Pedersen, Irene M

    2009-01-01

    , are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT...

  11. C2-domain containing calcium sensors in neuroendocrine secretion

    DEFF Research Database (Denmark)

    Pinheiro, Paulo S; Houy, Sébastien; Sørensen, Jakob B

    2016-01-01

    The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding to ca...... the properties and possible interplay of (some of) the major C2-domain containing calcium sensors in calcium-triggered exocytosis. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases"....

  12. [Expression of HIC-5/ARA55 in colonrectal cancer and its mechanisms of action].

    Science.gov (United States)

    Cui, Wei; Wang, Xin; Liu, Yu-cun; Wan, Yuan-lian; Guo, Hong-jie; Zhu, Jing

    2006-06-18

    To investigate the relationship between HIC-5 and colonrectal cancer. Samples were obtained from 42 patients with colonrectal cancer, and the mRNA and protein expressions of HIC-5/ARA55 were determined by RT-PCR and Western-blot respectively. FCM was used to investigate the apoptosis of Lovo in the HIC-5/ARA55 transfection cells. The expression of apoptosis related proteins Bax and Bcl-2 was determined by Western-blot, and the MTT assay was used to test cell growth speed affected by HIC-5/ARA55. The expression of HIC-5 significantly decreased on colonrectal cancer tissues as compared with adjacent normal tissues on mRNA level and protein level(Pcolonrectal cancer cells.

  13. The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins

    Directory of Open Access Journals (Sweden)

    Cesareni Gianni

    2008-07-01

    Full Text Available Abstract Background The human POB1/REPS2 (Partner of RalBP1 protein is highly conserved in mammals where it has been suggested to function as a molecular scaffold recruiting proteins involved in vesicular traffic and linking them to the actin cytoskeleton remodeling machinery. More recently POB1/REPS2 was found highly expressed in androgen-dependent prostate cancer cell lines, while one of its isoforms (isoform 2 is down regulated during prostate cancer progression. Results In this report we characterize the central proline rich domain of POB1/REPS2 and we describe for the first time its functional role in receptor endocytosis. We show that the ectopic expression of this domain has a dominant negative effect on the endocytosis of activated epidermal growth factor receptor (EGFR while leaving transferrin receptor endocytosis unaffected. By a combination of different approaches (phage display, bioinformatics predictions, peptide arrays, mutagenic analysis, in vivo co-immunoprecipitation, we have identified two closely spaced binding motifs for 14-3-3 and for the SH3 of the proteins Amphiphysin II and Grb2. Differently from wild type, proline rich domains that are altered in these motifs do not inhibit EGFR endocytosis, suggesting that these binding motifs play a functional role in this process. Conclusion Our findings are relevant to the characterization of the molecular mechanism underlying the involvement of POB1/REPS2, SH3 and 14-3-3 proteins in receptor endocytosis, suggesting that 14-3-3 could work by bridging the EGF receptor and the scaffold protein POB1/REPS2.

  14. The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation*

    Science.gov (United States)

    Chen, Yu-tsung Shane; Wu, Jianhong; Modrich, Paul; Hsieh, Tao-shih

    2016-01-01

    Eukaryotic topoisomerase 2 (Top2) and one of its interacting partners, topoisomerase IIβ binding protein 1 (TopBP1) are two proteins performing essential cellular functions. We mapped the interacting domains of these two proteins using co-immunoprecipitation and pulldown experiments with truncated or mutant Drosophila Top2 with various Ser-to-Ala substitutions. We discovered that the last 20 amino acids of Top2 represent the key region for binding with Mus101 (the Drosophila homolog of TopBP1) and that phosphorylation of Ser-1428 and Ser-1443 is important for Top2 to interact with the N terminus of Mus101, which contains the BRCT1/2 domains. The interaction between Mus101 and the Top2 C-terminal regulatory domain is phosphorylation-dependent because treatment with phosphatase abolishes their association in pulldown assays. The binding affinity of N-terminal Mus101 with a synthetic phosphorylated peptide spanning the last 25 amino acids of Top2 (with Ser(P)-1428 and Ser(P)-1443) was determined by surface plasmon resonance with a Kd of 0.57 μm. In an in vitro decatenation assay, Mus101 can specifically reduce the decatenation activity of Top2, and dephosphorylation of Top2 attenuates this response. Next, we endeavored to establish a cellular system for testing the biological function of Top2-Mus101 interaction. Top2-silenced S2 cells rescued by Top2Δ20, Top2 with 20 amino acids truncated from the C terminus, developed abnormally high chromosome numbers, which implies that Top2-Mus101 interaction is important for maintaining the fidelity of chromosome segregation during mitosis. PMID:27129233

  15. Structure function relations in PDZ-domain-containing proteins ...

    Indian Academy of Sciences (India)

    PDZ domains are subject toseveral means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to theeffect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical andbiophysical basis for this selectivity as well as promiscuity ...

  16. CBS domain-containing proteins are Rhizopus oryzae ferrioxamine receptors

    Science.gov (United States)

    Background: Iron-overload patients treated with deferoxamine are uniquely susceptible to mucormycosis, because Rhizopus spp. can obtain iron from ferrioxamine (deferoxamine + Fe**3+). Previously we have identified two closely related, ferrioxamine-inducible R. oryzae genes (FOB1 and FOB2) in which ...

  17. Plant SET domain-containing proteins: structure, function and regulation

    Czech Academy of Sciences Publication Activity Database

    Ng, D.W.K.; Wang, T.; Chandrasekharan, M.B.; Aramayo, R.; Kertbundit, Sunee; Hall, T.C.

    2007-01-01

    Roč. 1769, 5-6 (2007), s. 316-329 ISSN 0167-4781 Institutional research plan: CEZ:AV0Z50380511 Keywords : arabidopsis SET genes * alternative splicing * epigenetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.704, year: 2007

  18. diffHic: a Bioconductor package to detect differential genomic interactions in Hi-C data.

    Science.gov (United States)

    Lun, Aaron T L; Smyth, Gordon K

    2015-08-19

    Chromatin conformation capture with high-throughput sequencing (Hi-C) is a technique that measures the in vivo intensity of interactions between all pairs of loci in the genome. Most conventional analyses of Hi-C data focus on the detection of statistically significant interactions. However, an alternative strategy involves identifying significant changes in the interaction intensity (i.e., differential interactions) between two or more biological conditions. This is more statistically rigorous and may provide more biologically relevant results. Here, we present the diffHic software package for the detection of differential interactions from Hi-C data. diffHic provides methods for read pair alignment and processing, counting into bin pairs, filtering out low-abundance events and normalization of trended or CNV-driven biases. It uses the statistical framework of the edgeR package to model biological variability and to test for significant differences between conditions. Several options for the visualization of results are also included. The use of diffHic is demonstrated with real Hi-C data sets. Performance against existing methods is also evaluated with simulated data. On real data, diffHic is able to successfully detect interactions with significant differences in intensity between biological conditions. It also compares favourably to existing software tools on simulated data sets. These results suggest that diffHic is a viable approach for differential analyses of Hi-C data.

  19. A Toll/IL-1R/resistance domain-containing thioredoxin regulates phagocytosis in Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Mancilla-Herrera Ismael

    2012-10-01

    Full Text Available Abstract Background Entamoeba histolytica is a protozoan parasite that infects humans and causes amebiasis affecting developing countries. Phagocytosis of epithelial cells, erythrocytes, leucocytes, and commensal microbiota bacteria is a major pathogenic mechanism used by this parasite. A Toll/IL-1R/Resistance (TIR domain-containing protein is required in phagocytosis in the social ameba Dictyostelium discoideum, an ameba closely related to Entamoeba histolytica in phylogeny. In insects and vertebrates, TIR domain-containing proteins regulate phagocytic and cell activation. Therefore, we investigated whether E. histolytica expresses TIR domain-containing molecules that may be involved in the phagocytosis of erythrocytes and bacteria. Methods Using in silico analysis we explored in Entamoeba histolytica databases for TIR domain containing sequences. After silencing TIR domain containing sequences in trophozoites by siRNA we evaluated phagocytosis of erythrocytes and bacteria. Results We identified an E. histolytica thioredoxin containing a TIR-like domain. The secondary and tertiary structure of this sequence exhibited structural similarity to TIR domain family. Thioredoxin transcripts silenced in E. histolytica trophozoites decreased erythrocytes and E. coli phagocytosis. Conclusion TIR domain-containing thioredoxin of E. histolytica could be an important element in erythrocytes and bacteria phagocytosis.

  20. Ovarian Tumor (OTU)-domain Containing Viral Proteases Evade Ubiquitin- and ISG15-dependent Innate Immune Responses

    Science.gov (United States)

    Frias-Staheli, Natalia; Giannakopoulos, Nadia V.; Kikkert, Marjolein; Taylor, Shannon L.; Bridgen, Anne; Paragas, Jason J.; Richt, Juergen A.; Rowland, Raymond R.; Schmaljohn, Connie S.; Lenschow, Deborah J.; Snijder, Eric J.; García-Sastre, Adolfo; Virgin, Herbert Whiting

    2007-01-01

    Summary Ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) reversibly conjugate to proteins via a conserved LRLRGG C-terminal motif, mediating important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases of nairoviruses and arteriviruses hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. The biological significance of this activity of viral OTU domain-containing proteases was evidenced by their capacity to inhibit NF-κB dependent signaling and to antagonize the antiviral effects of ISG15 during Sindbis virus infection in vivo. The deconjugating activity of viral OTU proteases represents a novel viral immune evasion mechanism that inhibits Ub-and ISG15-dependent antiviral pathways. PMID:18078692

  1. HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel G; Pandey, Deo P; Jaskolska, Milena

    2009-01-01

    Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are protein...... a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.......Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins....... Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces...

  2. Coronal Heating Observed with Hi-C

    Science.gov (United States)

    Winebarger, Amy R.

    2013-01-01

    The recent launch of the High-Resolution Coronal Imager (Hi-C) as a sounding rocket has offered a new, different view of the Sun. With approx 0.3" resolution and 5 second cadence, Hi-C reveals dynamic, small-scale structure within a complicated active region, including coronal braiding, reconnection regions, Alfven waves, and flows along active region fans. By combining the Hi-C data with other available data, we have compiled a rich data set that can be used to address many outstanding questions in solar physics. Though the Hi-C rocket flight was short (only 5 minutes), the added insight of the small-scale structure gained from the Hi-C data allows us to look at this active region and other active regions with new understanding. In this talk, I will review the first results from the Hi-C sounding rocket and discuss the impact of these results on the coronal heating problem.

  3. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-01-01

    The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.

  4. The value of HIC1 and SIRT1 expression levels in thyroid nodule for assessing benign or malignant nodules

    Directory of Open Access Journals (Sweden)

    Chuan-Hua Fang

    2016-09-01

    Full Text Available Objective: To study the value of HIC1 and SIRT1 expression levels in thyroid nodule for assessing benign or malignant nodules. Method: 70 cases of surgically removed thyroid cancer tissue samples were collected as pathology group, 70 cases of corresponding para-carcinoma tissue specimens were collected as control group, immunohistochemistry was used to detect HIC1 and SIRT1 protein expression levels, and fluorescence quantitative PCR was used to detect the mRNA expression levels of HIC1 and SIRT1 as well as OXTR, CDH1, RASSF1A, TIMP3, MMP13, S100A4, CCNG2 and MK. Results: HIC1 expression levels in thyroid carcinoma tissue of pathology group were significantly lower than those of control group while SIRT1 expression levels were significantly higher than those of control group; compared with thyroid cancer tissue with TNM I-II stage, negative ductal infiltration and negative lymph node metastasis, HIC1 expression levels significantly decreased while SIRT1 expression levels significantly increased in thyroid cancer tissue with TNM III-IV stage, positive ductal infiltration and positive lymph node metastasis; in thyroid cancer tissue with positive HIC1 expression, OXTR, CDH1, RASSF1A and TIMP3 expression levels were significantly higher than those in thyroid cancer tissue with negative HIC1 expression; in thyroid cancer tissue with positive SIRT1 expression, MMP13, S100A4, CCNG2 and MK expression levels were significantly higher than those in thyroid cancer tissue with negative SIRT1 expression. Conclusion: HIC1 expression deletion and SIRT1 expression increase in thyroid nodule tissue are associated with the occurrence and development of thyroid carcinoma, and methylation and deacetylation may be epigenetic mechanism of HIC1 and SIRT1 to regulate cell proliferation, invasion and angiogenesis.

  5. Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5.

    Directory of Open Access Journals (Sweden)

    Michael Popp

    Full Text Available Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5, a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice.

  6. A geometric multigrid Poisson solver for domains containing solid inclusions

    Science.gov (United States)

    Botto, Lorenzo

    2013-03-01

    A Cartesian grid method for the fast solution of the Poisson equation in three-dimensional domains with embedded solid inclusions is presented and its performance analyzed. The efficiency of the method, which assume Neumann conditions at the immersed boundaries, is comparable to that of a multigrid method for regular domains. The method is light in terms of memory usage, and easily adaptable to parallel architectures. Tests with random and ordered arrays of solid inclusions, including spheres and ellipsoids, demonstrate smooth convergence of the residual for small separation between the inclusion surfaces. This feature is important, for instance, in simulations of nearly-touching finite-size particles. The implementation of the method, “MG-Inc”, is available online. Catalogue identifier: AEOE_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOE_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 19068 No. of bytes in distributed program, including test data, etc.: 215118 Distribution format: tar.gz Programming language: C++ (fully tested with GNU GCC compiler). Computer: Any machine supporting standard C++ compiler. Operating system: Any OS supporting standard C++ compiler. RAM: About 150MB for 1283 resolution Classification: 4.3. Nature of problem: Poisson equation in domains containing inclusions; Neumann boundary conditions at immersed boundaries. Solution method: Geometric multigrid with finite-volume discretization. Restrictions: Stair-case representation of the immersed boundaries. Running time: Typically a fraction of a minute for 1283 resolution.

  7. The Popeye Domain Containing Genes and Their Function in Striated Muscle

    Science.gov (United States)

    Schindler, Roland F. R.; Scotton, Chiara; French, Vanessa; Ferlini, Alessandra; Brand, Thomas

    2016-01-01

    The Popeye domain containing (POPDC) genes encode a novel class of cAMP effector proteins, which are abundantly expressed in heart and skeletal muscle. Here, we will review their role in striated muscle as deduced from work in cell and animal models and the recent analysis of patients carrying a missense mutation in POPDC1. Evidence suggests that POPDC proteins control membrane trafficking of interacting proteins. Furthermore, we will discuss the current catalogue of established protein-protein interactions. In recent years, the number of POPDC-interacting proteins has been rising and currently includes ion channels (TREK-1), sarcolemma-associated proteins serving functions in mechanical stability (dystrophin), compartmentalization (caveolin 3), scaffolding (ZO-1), trafficking (NDRG4, VAMP2/3) and repair (dysferlin) or acting as a guanine nucleotide exchange factor for Rho-family GTPases (GEFT). Recent evidence suggests that POPDC proteins might also control the cellular level of the nuclear proto-oncoprotein c-Myc. These data suggest that this family of cAMP-binding proteins probably serves multiple roles in striated muscle. PMID:27347491

  8. HIC1 interacts with a specific subunit of SWI/SNF complexes, ARID1A/BAF250A

    International Nuclear Information System (INIS)

    Van Rechem, Capucine; Boulay, Gaylor; Leprince, Dominique

    2009-01-01

    HIC1, a tumor suppressor gene epigenetically silenced in many human cancers encodes a transcriptional repressor involved in regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. HIC1 is also implicated in growth control since it recruits BRG1, one of the two alternative ATPases (BRM or BRG1) of SWI/SNF chromatin-remodeling complexes to repress transcription of E2F1 in quiescent fibroblasts. Here, through yeast two-hybrid screening, we identify ARID1A/BAF250A, as a new HIC1 partner. ARID1A/BAF250A is one of the two mutually exclusive ARID1-containing subunits of SWI/SNF complexes which define subsets of complexes endowed with anti-proliferative properties. Co-immunoprecipitation assays in WI38 fibroblasts and in BRG1-/- SW13 cells showed that endogenous HIC1 and ARID1A proteins interact in a BRG1-dependent manner. Furthermore, we demonstrate that HIC1 does not interact with BRM. Finally, sequential chromatin immunoprecipitation (ChIP-reChIP) experiments demonstrated that HIC1 represses E2F1 through the recruitment of anti-proliferative SWI/SNF complexes containing ARID1A.

  9. Dynamic Moss Observed with Hi-C

    Science.gov (United States)

    Alexander, Caroline; Winebarger, Amy; Morton, Richard; Savage, Sabrina

    2014-01-01

    The High-resolution Coronal Imager (Hi-C), flown on 11 July 2012, has revealed an unprecedented level of detail and substructure within the solar corona. Hi-C imaged a large active region (AR11520) with 0.2-0.3'' spatial resolution and 5.5s cadence over a 5 minute period. An additional dataset with a smaller FOV, the same resolution, but with a higher temporal cadence (1s) was also taken during the rocket flight. This dataset was centered on a large patch of 'moss' emission that initially seemed to show very little variability. Image processing revealed this region to be much more dynamic than first thought with numerous bright and dark features observed to appear, move and disappear over the 5 minute observation. Moss is thought to be emission from the upper transition region component of hot loops so studying its dynamics and the relation between the bright/dark features and underlying magnetic features is important to tie the interaction of the different atmospheric layers together. Hi-C allows us to study the coronal emission of the moss at the smallest scales while data from SDO/AIA and HMI is used to give information on these structures at different heights/temperatures. Using the high temporal and spatial resolution of Hi-C the observed moss features were tracked and the distribution of displacements, speeds, and sizes were measured. This allows us to comment on both the physical processes occurring within the dynamic moss and the scales at which these changes are occurring.

  10. [Research advances in CKLF-like MARVEL transmembrane domain containing member 5].

    Science.gov (United States)

    Yuan, Ye-qing; Xiao, Yun-bei; Liu, Zhen-hua; Zhang, Xiao-wei; Xu, Tao; Wang, Xiao-feng

    2012-12-01

    CKLF-like MARVEL transmembrane domain containing member(CMTM)is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM5 belongs to this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily(TM4SF). CMTM5 is broadly expressed in normal adult and fetal human tissues, but is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM5 may inhibit the proliferation, migration, and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear, CMTM5 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM5 may be a new target in the gene therapies for tumors, while further studies on CMTM5 and its anti-tumor mechanisms are warranted.

  11. A LysM Domain-Containing Gene OsEMSA1 Involved in Embryo sac Development in Rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Qian Zhu

    2017-09-01

    Full Text Available The embryo sac plays a vital role in sexual reproduction of angiosperms. LysM domain containing proteins with multiple lysin motifs are widespread proteins and are involved in plant defense responses against fungal chitins and bacterial peptidoglycans. Various studies have reported the role of LysM domain-containing proteins in plant defense mechanisms but their involvement in sexual reproduction remains largely unknown. Here, we report the involvement of a LysM domain-containing gene, EMBRYO SAC 1 (OsEMSA1, in the sexual reproduction of rice. The gene encoded a LysM domain-containing protein that was necessary for embryo sac development and function. The gene was expressed in root, stem, leaf tissues, panicle and ovaries and had some putative role in hormone regulation. Suppression of OsEMSA1 expression resulted in a defective embryo sac with poor differentiation of gametophytic cells, which consequently failed to attract pollen tubes and so reduced the panicle seed-setting rate. Our data offers new insight into the functions of LysM domain-containing proteins in rice.

  12. Loop Evolution Observed with AIA and Hi-C

    Science.gov (United States)

    Mulu-Moore, Fana; Winebarger, Amy R.; Cirtain, Jonathan W.; Kobayashi, Ken; Korreck, Kelly E.; Golub, Leon; Kuzin, Sergei; Walsh, Robert William; DeForest, Craig E.; De Pontieu, Bart; hide

    2012-01-01

    In the past decade, the evolution of EUV loops has been used to infer the loop substructure. With the recent launch of High Resolution Coronal Imager (Hi-C), this inference can be validated. In this presentation we discuss the first results of loop analysis comparing AIA and Hi-C data. In the past decade, the evolution of EUV loops has been used to infer the loop substructure. With the recent launch of High Resolution Coronal Imager (Hi-C), this inference can be validated. In this presentation we discuss the first results of loop analysis comparing AIA and Hi-C data.

  13. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    Science.gov (United States)

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Effects of abhydrolase domain containing 5 gene (ABHD5) expression and variations on chicken fat metabolism.

    Science.gov (United States)

    Ouyang, Hongjia; Liu, Qing; Xu, Jiguo; Zeng, Fang; Pang, Xiaolin; Jebessa, Endashaw; Liang, Shaodong; Nie, Qinghua; Zhang, Xiquan

    2016-01-01

    Abhydrolase domain containing 5 gene (ABHD5), also known as comparative gene identification 58 (CGI-58), is a member of the α/β-hydrolase family as a protein cofactor of ATGL stimulating its triacylglycerol hydrolase activity. In this study, we aim to characterize the expression and variations of ABHD5 and to study their functions in chicken fat metabolism. We compared the ABHD5 expression level in various tissues and under different nutrition conditions, identified the variations of ABHD5, and associated them with production traits in an F2 resource population of chickens. Overexpression analysis with two different genotypes and siRNA interfering analysis of ABHD5 were performed in chicken preadipocytes. Chicken ABDH5 was expressed widely and most predominantly in adipose tissue. Five SNPs of the ABHD5 gene were identified and genotyped in the F2 resource population. The c.490C > T SNP was associated with subcutaneous fat thickness (P  C SNP was also associated with chicken body weight (P chicken preadipocytes, overexpression of wild type ABDH5 did not affect the mRNA level of ATGL (adipose triglyceride lipase) but markedly decreased (P chickens with a high fat diet. These results suggest that expression and variations of ABHD5 may affect fat metabolism through regulating the activity of ATGL in chickens. © 2015 Poultry Science Association Inc.

  15. Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli.

    Science.gov (United States)

    Xiao, Wenjun; Jiang, Li; Wang, Weiyu; Wang, Ruyue; Fan, Jun

    2018-02-01

    Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Structural Evaluation on HIC Transport Packaging under Accident Conditions

    International Nuclear Information System (INIS)

    Chung, Sung Hwan; Kim, Duck Hoi; Jung, Jin Se; Yang, Ke Hyung; Lee, Heung Young

    2005-01-01

    HIC transport packaging to transport a high integrity container(HIC) containing dry spent resin generated from nuclear power plants is to comply with the regulatory requirements of Korea and IAEA for Type B packaging due to the high radioactivity of the content, and to maintain the structural integrity under normal and accident conditions. It must withstand 9 m free drop impact onto an unyielding surface and 1 m drop impact onto a mild steel bar in a position causing maximum damage. For the conceptual design of a cylindrical HIC transport package, three dimensional dynamic structural analysis to ensure that the integrity of the package is maintained under all credible loads for 9 m free drop and 1 m puncture conditions were carried out using ABAQUS code.

  18. Hi-C Observations of Penumbral Bright Dots

    Science.gov (United States)

    Alpert, S. E.; Tiwari, S. K.; Moore, R. L.; Savage, S. L.; Winebarger, A. R.

    2014-01-01

    We use high-quality data obtained by the High Resolution Coronal Imager (Hi-C) to examine bright dots (BDs) in a sunspot's penumbra. The sizes of these BDs are on the order of 1 arcsecond (1") and are therefore hard to identify using the Atmospheric Imaging Assembly's (AIA) 0.6" pixel(exp -1) resolution. These BD become readily apparent with Hi-C's 0.1" pixel(exp -1) resolution. Tian et al. (2014) found penumbral BDs in the transition region (TR) by using the Interface Region Imaging Spectrograph (IRIS). However, only a few of their dots could be associated with any enhanced brightness in AIA channels. In this work, we examine the characteristics of the penumbral BDs observed by Hi-C in a sunspot penumbra, including their sizes, lifetimes, speeds, and intensity. We also attempt to find any association of these BDs to the IRIS BDs. There are fewer Hi-C BDs in the penumbra than seen by IRIS, though different sunspots were studied. We use 193 Angstroms Hi-C data from July 11, 2012 which observed from approximately 18:52:00 UT- 18:56:00 UT and supplement it with data from AIA's 193 Angstrom passband to see the complete lifetime of the dots that were born before and/or lasted longer than Hi- C's 5-minute observation period. We use additional AIA passbands and compare the light curves of the BDs at different temperatures to test whether the Hi-C BDs are TR BDs. We find that most Hi-C BDs show clear movement, and of those that do, they move in a radial direction, toward or away from the sunspot umbra. Single BDs interact with other BDs, combining to fade away or brighten. The BDs that do not interact with other BDs tend to move less. Many of the properties of our BDs are similar to the extreme values of the IRIS BDs, e.g., they move slower on average and their sizes and lifetimes are on the higher end of the IRIS BDs. We infer that our penumbral BDs are the large-scale end of the distribution of BDs observed by IRIS.

  19. Hi-C OBSERVATIONS OF SUNSPOT PENUMBRAL BRIGHT DOTS

    Energy Technology Data Exchange (ETDEWEB)

    Alpert, Shane E. [Department of Physics and Astronomy, Rice University, Houston, TX, 77005 (United States); Tiwari, Sanjiv K.; Moore, Ronald L.; Winebarger, Amy R.; Savage, Sabrina L. [NASA Marshall Space Flight Center, ZP 13, Huntsville, AL 35812 (United States)

    2016-05-01

    We report observations of bright dots (BDs) in a sunspot penumbra using High Resolution Coronal Imager (Hi-C) data in 193 Å and examine their sizes, lifetimes, speeds, and intensities. The sizes of the BDs are on the order of 1″ and are therefore hard to identify in the Atmospheric Imaging Assembly (AIA) 193 Å images, which have a 1.″2 spatial resolution, but become readily apparent with Hi-C's spatial resolution, which is five times better. We supplement Hi-C data with data from AIA's 193 Å passband to see the complete lifetime of the BDs that appeared before and/or lasted longer than Hi-C's three-minute observation period. Most Hi-C BDs show clear lateral movement along penumbral striations, either toward or away from the sunspot umbra. Single BDs often interact with other BDs, combining to fade away or brighten. The BDs that do not interact with other BDs tend to have smaller displacements. These BDs are about as numerous but move slower on average than Interface Region Imaging Spectrograph (IRIS) BDs, which was recently reported by Tian et al., and the sizes and lifetimes are on the higher end of the distribution of IRIS BDs. Using additional AIA passbands, we compare the light curves of the BDs to test whether the Hi-C BDs have transition region (TR) temperatures like those of the IRIS BDs. The light curves of most Hi-C BDs peak together in different AIA channels, indicating that their temperatures are likely in the range of the cooler TR (1−4 × 10{sup 5} K).

  20. Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5

    Directory of Open Access Journals (Sweden)

    Jonathan M. Peterson

    2014-09-01

    Full Text Available Metabolic dysfunction in skeletal muscle is a major contributor to the development of type 2 diabetes. Endurance exercise training has long been established as an effective means to directly restore skeletal muscle glucose and lipid uptake and metabolism. However, in addition to the direct effects of skeletal muscle on glucose and lipids, there is renewed interest in the ability of skeletal muscle to coordinate metabolic activity of other tissues, such as adipose tissue and liver. The purpose of this study was to examine the effects of endurance exercise on the expression level of two novel muscle-derived secreted factors, or myokines, Myonectin and Fibronectin type III domain containing 5 (FNDC5, the precursor for Irisin.Methods. We performed immunoblot analysis and quantitative real-time PCR analysis of Myonectin and FNDC5 in the diaphragm muscles of obese Zucker rat (OZR and lean Zucker rat (LZR with 9 weeks of aerobic training on a motorized treadmill.Results. We show that myonectin gene expression is increased in the OZR model of obesity and decreases with exercise in both lean and obese Zucker rats. Conversely, myonectin protein concentration was elevated with exercise. Similarly, FNDC5 mRNA levels are significantly higher in the OZR, however exercise training had no effect on the expression level of FNDC5 in either the LZR or OZR. We did not observe any difference in muscle protein content of Irisin with obesity or exercise.Conclusion. Our data shows that exercise training does not increase either FNDC5 or myonectin gene expression, indicating that increased transcriptional regulation of these myokines is not induced by exercise. However, our data also indicates a yet to be explored disconnect between myonectin gene expression and protein content. Further, this report highlights the importance of verifying reference genes when completing gene expression analysis. We found that many commonly used reference genes varied significantly by

  1. Glucocorticoid receptor binding to chromatin is selectively controlled by the coregulator Hic-5 and chromatin remodeling enzymes.

    Science.gov (United States)

    Lee, Brian H; Stallcup, Michael R

    2017-06-02

    The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress pathways by binding to genomic regulatory elements of target genes and recruiting coregulator proteins to remodel chromatin and regulate transcription complex assembly. The coregulator hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid (GC) regulation of some genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to some genes but not others is unclear. Here we show that site-specific blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes but not for other GC-regulated genes. Furthermore, CHD9 and BRM were required for GR occupancy and chromatin remodeling at GR-binding regions associated with blocked genes but not at GR-binding regions associated with other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites associated with blocked genes. Thus, Hic-5 regulates GR binding site selection by a novel mechanism, exploiting gene-specific requirements for chromatin remodeling enzymes to selectively influence DNA occupancy and gene regulation by a transcription factor. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Coronal Loop Evolution Observed with AIA and Hi-C

    Science.gov (United States)

    Mulu-Moore, Fana; Winebarger, A.; Cirtain, J.; Kobayashi, K.; Korreck, K.; Golub, L.; Kuzin. S.; Walsh, R.; DeForest, C.; DePontieu, B.; hide

    2012-01-01

    Despite much progress toward understanding the dynamics of the solar corona, the physical properties of coronal loops are not yet fully understood. Recent investigations and observations from different instruments have yielded contradictory results about the true physical properties of coronal loops. In the past, the evolution of loops has been used to infer the loop substructure. With the recent launch of High Resolution Coronal Imager (Hi-C), this inference can be validated. In this poster we discuss the first results of loop analysis comparing AIA and Hi-C data. We find signatures of cooling in a pixel selected along a loop structure in the AIA multi-filter observations. However, unlike previous studies, we find that the cooling time is much longer than the draining time. This is inconsistent with previous cooling models.

  3. Effect of Microstructure on HIC Susceptibility of API X70MS Linepipe Steel

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Joon-Ho; Sim, Ho-Seop; Park, Byung-Gyu [Dongkuk Steel R and D Center, Pohang (Korea, Republic of); Cho, Kyung-Mox [Pusan National University, Busan (Korea, Republic of)

    2017-06-15

    The resistance of hydrogen induced cracking (HIC) was investigated with different microstructures of API X70MS steel. Ferrite/acicular ferrite (F/AF), deformed ferrite/acicular ferrite, ferrite/bainite (F/B) and single acicular ferrite (AF) were obtained by thermo-mechanical controlled process (TMCP) with changing rolling and cooling conditions. HIC resistance was found to be affected by the type as well as morphology of the microstructure, and thus the behavior of crack initiation and propagation could be analyzed. It was found that single AF and deformed F/AF with uniformly distributed dislocation reduced HIC initiation. Banded microstructure with a hardness value below 250 HV, such as AF, showed good HIC propagation resistance due to high toughness. AF generally exhibited excellent crack initiation and propagation resistance, namely the best HIC susceptibility performance. AF might redeem the HIC resistance for the banded microstructure also.

  4. Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

    Science.gov (United States)

    Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

    2016-01-01

    CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

  5. Research Advances in CKLFSF-like MARVEL Transmembrane Domain Containing Member 3.

    Science.gov (United States)

    Hu, Feng-zhan; Sheng, Zheng-zuo; Qin, Cai-peng; Xu, Tao

    2016-06-10

    CKLF-like MARVEL transmembrane domain containing member/chemokine-like factor super family member (CKLFSF/CMTM) is a novel tumor suppressor gene. CMTM3 is broadly expressed in normal human tissues and evolutionary conserved,especially in testis,spleen,and some cells of peripheral blood mononuclear cells. However,its expression is undetectable or down-regulated in most carcinoma cell lines and tissues. Restoration of CMTM3 may inhibit the proliferation,migration,and invasion of carcinoma cells. Although the exact mechanism of its anti-tumor activity remains unclear,CKLFSF3/CMTM3 is closely connected with immune system and associated with sex during tumorigenesis. The study advances of CKLFSF3/CMTM3 are elaborated in this review as CMTM3 may be a new target in the gene therapies for tumors,especially genitourinary tumors,while further studies on CMTM3 and its anti-tumor mechanisms are warranted.

  6. Investigate Global Chromosomal Interaction by Hi-C in Human Naive CD4 T Cells.

    Science.gov (United States)

    Meng, Xiangzhi; Riley, Nicole; Thompson, Ryan; Sharma, Siddhartha

    2018-01-01

    Hi-C is a methodology developed to reveal chromosomal interactions from a genome-wide perspective. Here, we described a protocol for generating Hi-C sequencing libraries in resting and activated human naive CD4 T cells to investigate activation-induced chromatin structure re-arrangement in T cell activation followed by a section reviewing the general concepts of Hi-C data analysis.

  7. The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development

    Science.gov (United States)

    Kirchmaier, Bettina C.; Poon, Kar Lai; Schwerte, Thorsten; Huisken, Jan; Winkler, Christoph; Jungblut, Benno; Stainier, Didier Y.; Brand, Thomas

    2013-01-01

    The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)s878) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system. PMID:22290329

  8. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

    Science.gov (United States)

    Bhoj, Elizabeth J.; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J.; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B.; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A. Schrier; Tarnopolsky, Mark A.; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-01-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

  9. Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

    Energy Technology Data Exchange (ETDEWEB)

    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Eizuru, Yoshito [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2010-06-04

    Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.

  10. I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

    Energy Technology Data Exchange (ETDEWEB)

    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Yoshimitsu, Makoto; Hachiman, Miho [Division of Hematology and Immunology, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ikeda, Masanori [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2015-12-15

    The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.

  11. HIC1 Expression Distinguishes Intestinal Carcinomas Sensitive to Chemotherapy

    Czech Academy of Sciences Publication Activity Database

    Janečková, Lucie; Kolář, Michal; Švec, Jiří; Láníková, Lucie; Pospíchalová, Vendula; Baloghová, Nikol; Vojtěchová, Martina; Šloncová, Eva; Strnad, Hynek; Kořínek, Vladimír

    2016-01-01

    Roč. 9, č. 2 (2016), s. 99-107 ISSN 1944-7124 R&D Projects: GA ČR GAP305/12/2347; GA MŠk LO1419; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : direct target gene * cancer 1 hic1 * colorectal - cancer * breast- cancer * lung- cancer * tgf-beta * methylation * hypermethylation * subtypes * cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.077, year: 2015

  12. Discovery of Finely Structured Dynamic Solar Corona Observed in the Hi-C Telescope

    Science.gov (United States)

    Winebarger, A.; Cirtain, J.; Golub, L.; DeLuca, E.; Savage, S.; Alexander, C.; Schuler, T.

    2014-01-01

    In the summer of 2012, the High-resolution Coronal Imager (Hi-C) flew aboard a NASA sounding rocket and collected the highest spatial resolution images ever obtained of the solar corona. One of the goals of the Hi-C flight was to characterize the substructure of the solar corona. We therefore examine how the intensity scales from AIA resolution to Hi-C resolution. For each low-resolution pixel, we calculate the standard deviation in the contributing high-resolution pixel intensities and compare that to the expected standard deviation calculated from the noise. If these numbers are approximately equal, the corona can be assumed to be smoothly varying, i.e. have no evidence of substructure in the Hi-C image to within Hi-C's ability to measure it given its throughput and readout noise. A standard deviation much larger than the noise value indicates the presence of substructure. We calculate these values for each low-resolution pixel for each frame of the Hi-C data. On average, 70 percent of the pixels in each Hi-C image show no evidence of substructure. The locations where substructure is prevalent is in the moss regions and in regions of sheared magnetic field. We also find that the level of substructure varies significantly over the roughly 160 s of the Hi-C data analyzed here. This result indicates that the finely structured corona is concentrated in regions of heating and is highly time dependent.

  13. Coronal Fine Structure in Dynamic Events Observed by Hi-C

    Science.gov (United States)

    Winebarger, Amy; Schuler, Timothy

    2013-01-01

    The High-Resolution Coronal Imager (Hi-C) flew aboard a NASA sounding rocket on 2012 July 11 and captured roughly 345 s of high spatial and temporal resolution images of the solar corona in a narrowband 193 Angstrom channel. We have analyzed the fluctuations in intensity of Active Region 11520. We selected events based on a lifetime greater than 11 s (two Hi-C frames) and intensities greater than a threshold determined from the photon and readout noise. We compare the Hi-C events with those determined from AIA. We find that HI-C detects shorter and smaller events than AIA. We also find that the intensity increase in the Hi-C events is approx. 3 times greater than the intensity increase in the AIA events we conclude the events are related to linear sub-structure that is unresolved by AIA

  14. Enhancing Hi-C data resolution with deep convolutional neural network HiCPlus.

    Science.gov (United States)

    Zhang, Yan; An, Lin; Xu, Jie; Zhang, Bo; Zheng, W Jim; Hu, Ming; Tang, Jijun; Yue, Feng

    2018-02-21

    Although Hi-C technology is one of the most popular tools for studying 3D genome organization, due to sequencing cost, the resolution of most Hi-C datasets are coarse and cannot be used to link distal regulatory elements to their target genes. Here we develop HiCPlus, a computational approach based on deep convolutional neural network, to infer high-resolution Hi-C interaction matrices from low-resolution Hi-C data. We demonstrate that HiCPlus can impute interaction matrices highly similar to the original ones, while only using 1/16 of the original sequencing reads. We show that the models learned from one cell type can be applied to make predictions in other cell or tissue types. Our work not only provides a computational framework to enhance Hi-C data resolution but also reveals features underlying the formation of 3D chromatin interactions.

  15. Understanding spatial organizations of chromosomes via statistical analysis of Hi-C data

    Science.gov (United States)

    Hu, Ming; Deng, Ke; Qin, Zhaohui; Liu, Jun S.

    2015-01-01

    Understanding how chromosomes fold provides insights into the transcription regulation, hence, the functional state of the cell. Using the next generation sequencing technology, the recently developed Hi-C approach enables a global view of spatial chromatin organization in the nucleus, which substantially expands our knowledge about genome organization and function. However, due to multiple layers of biases, noises and uncertainties buried in the protocol of Hi-C experiments, analyzing and interpreting Hi-C data poses great challenges, and requires novel statistical methods to be developed. This article provides an overview of recent Hi-C studies and their impacts on biomedical research, describes major challenges in statistical analysis of Hi-C data, and discusses some perspectives for future research. PMID:26124977

  16. Preliminary Analysis of Radiation Shielding for B-type HIC Transport Package

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dohyung; Lee, Unjang; Ko, Jaehoon; Choi, Kyu-Sup [Korea Nuclear Engineering and Service Corporation, Seoul (Korea, Republic of)

    2007-10-15

    A radiation shielding analysis has been conducted using a computer program MCNP5 for a B-type HIC (High Integrated Container) Transport Package, which contains HIC with radioactive waste or Spent Resin, for transportation from nuclear power plant sites to disposal repository. Radiation source term is first carefully determined from the safety analysis reports related to HIC for appropriate calculation. And then MCNP (v.5) is performed to obtain the minimum thickness of HIC transport package, which meets the dose rate limit for HIC transport package prescribed in Korea Nuclear Law and IAEA Safety Standards for Radioactive Material Transport. In addition, some other analyses are done about the trend of dose rates depending on the thickness of shielding material and distance from the package.

  17. Popeye domain containing gene 2 (Popdc2) is a myocyte-specific differentiation marker during chick heart development.

    Science.gov (United States)

    Breher, Stephanie S; Mavridou, Eleftheria; Brenneis, Christian; Froese, Alexander; Arnold, Hans-Henning; Brand, Thomas

    2004-03-01

    The Popeye domain containing (popdc) genes constitute a novel gene family encoding proteins of the plasma membrane in muscle cells, with three N-terminal transmembrane domains and a cytoplasmic carboxy terminus. In vertebrates, three members of the Popdc gene family have been described. However, in the chick system only two cDNAs, Popdc1 and Popdc3, have been cloned previously. By screening a chick expressed sequence tag database, we report here the identification of five alternatively spliced chick Popdc2 cDNAs with different carboxy termini. Northern blot analysis revealed expression of Popdc2 predominantly in the myocardium and weaker expression in skeletal muscle. By whole-mount in situ hybridization, chick Popdc2 was first detected at Hamburger and Hamilton (HH) stage 7 within the anterior part of the heart fields. In the tubular heart, atrial and ventricular precursor cells stained positively for Popdc2. Weaker expression was observed in myocardium of the outflow tract and sinus venosus. By HH stage 18, the outer curvature myocardium was strongly stained, whereas expression in myocardium of the inner curvature was negligible. Popdc2 expression was absent from the endocardium and propepicardial organ. At HH stage 36, Popdc2 expression was confined to the compact layer myocardium. In addition to the heart, Popdc2 expression was also observed in the myotome and in the muscle-forming fields of the limbs. Our results indicate that Popdc2 is highly expressed in the developing heart and may serve as a novel marker of myocardial differentiation in the chick embryo. Copyright 2004 Wiley-Liss, Inc.

  18. Small activating RNA restores the activity of the tumor suppressor HIC-1 on breast cancer.

    Directory of Open Access Journals (Sweden)

    Feng Zhao

    Full Text Available HIC-1 is a gene that is hypermethylated in cancer, and commonly downregulated in human breast cancer. However, the precise mechanisms and molecular pathways regulated by HIC-1 remain unclear. We assessed HIC-1 expression on a tissue microarray containing 80 cases of breast cancer. We also analyzed its biological function by restoring HIC-1 expression using 5-aza-2' deoxycytidine (5-CdR and small-activating RNAs for the reversal of HIC-1 tumor suppressive effects on MCF-7 and MDA-MB-231 cell lines. An Agilent Q44h global expressing microarray was probed after restoring the expression of HIC-1. Data demonstrated that HIC-1 expression was reduced significantly in breast cancer tissues. HIC-1 immunohistochemistry resulted in mean staining scores in cancer tissue and normal ductal epithelia of 3.54 and 8.2, respectively (p<0.01. 5-CdR partially reversed HIC-1 expression, and modulated cell growth and apoptosis. dsHIC1-2998, an saRNA, showed activating efficacy in breast cancer cells. A group of differentially expressed genes were characterized by cDNA microarray. Upon saRNA treatment, genes upregulated included those involved in immune activation, cell cycle interference, the induction of apoptosis, anti-metastasis, and cell differentiation. Downregulated genes included oncogenes and those that play roles in cell invasion, cell growth, and cell division. Our findings may provide valuable resources not only for gene functional studies, but also for potential clinical applications to develop novel drug targets.

  19. Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

    Science.gov (United States)

    Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

    2017-06-02

    Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

  20. HiCUP: pipeline for mapping and processing Hi-C data.

    Science.gov (United States)

    Wingett, Steven; Ewels, Philip; Furlan-Magaril, Mayra; Nagano, Takashi; Schoenfelder, Stefan; Fraser, Peter; Andrews, Simon

    2015-01-01

    HiCUP is a pipeline for processing sequence data generated by Hi-C and Capture Hi-C (CHi-C) experiments, which are techniques used to investigate three-dimensional genomic organisation. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also produces an easy-to-interpret yet detailed quality control (QC) report that assists in refining experimental protocols for future studies. The software is freely available and has already been used for processing Hi-C and CHi-C data in several recently published peer-reviewed studies.

  1. GO JUP HIC HIGHRES ENERGETIC ION COUNT RATE V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This data set provides energetic (MeV) ion count rates and events measured by the Heavy Ion Counter (HIC) instrument on the Galileo spacecraft. These data are...

  2. GO JUP HIC DERIVED ENERGETIC ION COMPOSITION V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This data set provides energetic (MeV) ion fluxes for a variety of different Z values (carbon, oxygen, sulfur) derived from the Heavy Ion Counter (HIC) instrument on...

  3. Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation.

    Science.gov (United States)

    Belaghzal, Houda; Dekker, Job; Gibcus, Johan H

    2017-07-01

    Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Viral Interactions with PDZ Domain-Containing Proteins—An Oncogenic Trait?

    Directory of Open Access Journals (Sweden)

    Claire D. James

    2016-01-01

    Full Text Available Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9, encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ interaction modules. In many cases (but not always, the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis.

  5. Optimization and quality control of genome-wide Hi-C library preparation.

    Science.gov (United States)

    Zhang, Xiang-Yuan; He, Chao; Ye, Bing-Yu; Xie, De-Jian; Shi, Ming-Lei; Zhang, Yan; Shen, Wen-Long; Li, Ping; Zhao, Zhi-Hu

    2017-09-20

    Highest-throughput chromosome conformation capture (Hi-C) is one of the key assays for genome- wide chromatin interaction studies. It is a time-consuming process that involves many steps and many different kinds of reagents, consumables, and equipments. At present, the reproducibility is unsatisfactory. By optimizing the key steps of the Hi-C experiment, such as crosslinking, pretreatment of digestion, inactivation of restriction enzyme, and in situ ligation etc., we established a robust Hi-C procedure and prepared two biological replicates of Hi-C libraries from the GM12878 cells. After preliminary quality control by Sanger sequencing, the two replicates were high-throughput sequenced. The bioinformatics analysis of the raw sequencing data revealed the mapping-ability and pair-mate rate of the raw data were around 90% and 72%, respectively. Additionally, after removal of self-circular ligations and dangling-end products, more than 96% of the valid pairs were reached. Genome-wide interactome profiling shows clear topological associated domains (TADs), which is consistent with previous reports. Further correlation analysis showed that the two biological replicates strongly correlate with each other in terms of both bin coverage and all bin pairs. All these results indicated that the optimized Hi-C procedure is robust and stable, which will be very helpful for the wide applications of the Hi-C assay.

  6. A CH domain-containing N terminus in NuMA?

    Science.gov (United States)

    Novatchkova, Maria; Eisenhaber, Frank

    2002-10-01

    Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA-dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin-homology (CH) domain, a motif with binding activity for actin-like molecules. We substantiate the primary sequence analysis-based prediction with secondary structure and fold recognition analysis, and we propose the N-terminal CH domain of NuMA as a likely interaction site for actin-related protein 1 (Arp1) protein of the dynactin/dynein complex.

  7. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  8. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-01-01

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

  9. Localization and distribution offibrinogen C domain containing 1(FIBCD1) in human tissues

    DEFF Research Database (Denmark)

    von Huth, Sebastian; Møller, Jesper Bonnet; Schlosser, Anders

    2016-01-01

    in an immunohistochemistry-based analysis and demonstrate that FIBCD1 protein is highly expressed at the apical surfaces of the epithelium throughout the gastrointestinal tract, in the uterus, testis, bladder, gallbladder and the salivary glands. To a lesser extent, FIBCD1 is expressed in the pancreas, the spleen...

  10. Localization and distribution of fibrinogen C domain containing 1 (FIBCD1) in human tissues

    DEFF Research Database (Denmark)

    von Huth, Sebastian; Møller, Jesper Bonnet; Schlosser, Anders

    in an immunohistochemistry-based analysis and demonstrate that FIBCD1 protein is highly expressed at the apical surfaces of the epithelium throughout the gastrointestinal tract, in the uterus, testis, bladder, gallbladder and the salivary glands. To a lesser extent, FIBCD1 is expressed in the pancreas, the spleen...

  11. Comparative Hi-C Reveals that CTCF Underlies Evolution of Chromosomal Domain Architecture

    Directory of Open Access Journals (Sweden)

    Matteo Vietri Rudan

    2015-03-01

    Full Text Available Topological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF’s long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules.

  12. Inferential Structure Determination of Chromosomes from Single-Cell Hi-C Data

    Science.gov (United States)

    Nilges, Michael

    2016-01-01

    Chromosome conformation capture (3C) techniques have revealed many fascinating insights into the spatial organization of genomes. 3C methods typically provide information about chromosomal contacts in a large population of cells, which makes it difficult to draw conclusions about the three-dimensional organization of genomes in individual cells. Recently it became possible to study single cells with Hi-C, a genome-wide 3C variant, demonstrating a high cell-to-cell variability of genome organization. In principle, restraint-based modeling should allow us to infer the 3D structure of chromosomes from single-cell contact data, but suffers from the sparsity and low resolution of chromosomal contacts. To address these challenges, we adapt the Bayesian Inferential Structure Determination (ISD) framework, originally developed for NMR structure determination of proteins, to infer statistical ensembles of chromosome structures from single-cell data. Using ISD, we are able to compute structural error bars and estimate model parameters, thereby eliminating potential bias imposed by ad hoc parameter choices. We apply and compare different models for representing the chromatin fiber and for incorporating singe-cell contact information. Finally, we extend our approach to the analysis of diploid chromosome data. PMID:28027298

  13. Hospital Incident Command System (HICS) performance in Iran; decision making during disasters.

    Science.gov (United States)

    Djalali, Ahmadreza; Castren, Maaret; Hosseinijenab, Vahid; Khatib, Mahmoud; Ohlen, Gunnar; Kurland, Lisa

    2012-02-06

    Hospitals are cornerstones for health care in a community and must continue to function in the face of a disaster. The Hospital Incident Command System (HICS) is a method by which the hospital operates when an emergency is declared. Hospitals are often ill equipped to evaluate the strengths and vulnerabilities of their own management systems before the occurrence of an actual disaster. The main objective of this study was to measure the decision making performance according to HICS job actions sheets using tabletop exercises. This observational study was conducted between May 1st 2008 and August 31st 2009. Twenty three Iranian hospitals were included. A tabletop exercise was developed for each hospital which in turn was based on the highest probable risk. The job action sheets of the HICS were used as measurements of performance. Each indicator was considered as 1, 2 or 3 in accordance with the HICS. Fair performance was determined as hospitals had a hospital disaster management plan. The performance according to HICS was intermediate for 83% (n = 19) of the participating hospitals. No hospital had a high level of performance. The performance level for the individual sections was intermediate or fair, except for the logistic and finance sections which demonstrated a higher level of performance. The public hospitals had overall higher performances than university hospitals (P = 0.04). The decision making performance in the Iranian hospitals, as measured during table top exercises and using the indicators proposed by HICS was intermediate to poor. In addition, this study demonstrates that the HICS job action sheets can be used as a template for measuring the hospital response. Simulations can be used to assess preparedness, but the correlation with outcome remains to be studied.

  14. Hospital incident command system (HICS performance in Iran; decision making during disasters

    Directory of Open Access Journals (Sweden)

    Djalali Ahmadreza

    2012-02-01

    Full Text Available Abstract Background Hospitals are cornerstones for health care in a community and must continue to function in the face of a disaster. The Hospital Incident Command System (HICS is a method by which the hospital operates when an emergency is declared. Hospitals are often ill equipped to evaluate the strengths and vulnerabilities of their own management systems before the occurrence of an actual disaster. The main objective of this study was to measure the decision making performance according to HICS job actions sheets using tabletop exercises. Methods This observational study was conducted between May 1st 2008 and August 31st 2009. Twenty three Iranian hospitals were included. A tabletop exercise was developed for each hospital which in turn was based on the highest probable risk. The job action sheets of the HICS were used as measurements of performance. Each indicator was considered as 1, 2 or 3 in accordance with the HICS. Fair performance was determined as Results None of the participating hospitals had a hospital disaster management plan. The performance according to HICS was intermediate for 83% (n = 19 of the participating hospitals. No hospital had a high level of performance. The performance level for the individual sections was intermediate or fair, except for the logistic and finance sections which demonstrated a higher level of performance. The public hospitals had overall higher performances than university hospitals (P = 0.04. Conclusions The decision making performance in the Iranian hospitals, as measured during table top exercises and using the indicators proposed by HICS was intermediate to poor. In addition, this study demonstrates that the HICS job action sheets can be used as a template for measuring the hospital response. Simulations can be used to assess preparedness, but the correlation with outcome remains to be studied.

  15. Overexpression of a soybean nuclear localized type-III DnaJ domain-containing HSP40 reveals its roles in cell death and disease resistance.

    Science.gov (United States)

    Liu, Jian-Zhong; Whitham, Steven A

    2013-04-01

    Heat-shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co-chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full-length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type-III DnaJ domain-containing HSP40 (GmHSP40.1), a co-chaperone of HSP70, caused hypersensitive response (HR)-like cell death. The HR-like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP-GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR-inducing ability. GmHSP40.1 co-localized with HcRed-SE, a protein involved in pri-miRNA processing, which has been shown to be co-localized with SR33-YFP, a protein involved in pre-mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear-localized DnaJ domain-containing GmHSP40.1 in cell death and disease resistance in soybean. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

  16. OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes.

    Science.gov (United States)

    Vidal, Enrique; le Dily, François; Quilez, Javier; Stadhouders, Ralph; Cuartero, Yasmina; Graf, Thomas; Marti-Renom, Marc A; Beato, Miguel; Filion, Guillaume J

    2018-01-31

    The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb. © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. The Substructure of the Solar Corona Observed in the Hi-C Telescope

    Science.gov (United States)

    Winebarger, A.; Cirtain, J.; Golub, L.; DeLuca, E.; Savage, S.; Alexander, C.; Schuler, T.

    2014-01-01

    In the summer of 2012, the High-resolution Coronal Imager (Hi-C) flew aboard a NASA sounding rocket and collected the highest spatial resolution images ever obtained of the solar corona. One of the goals of the Hi-C flight was to characterize the substructure of the solar corona. We therefore calculate how the intensity scales from a low-resolution (AIA) pixels to high-resolution (Hi-C) pixels for both the dynamic events and "background" emission (meaning, the steady emission over the 5 minutes of data acquisition time). We find there is no evidence of substructure in the background corona; the intensity scales smoothly from low-resolution to high-resolution Hi-C pixels. In transient events, however, the intensity observed with Hi-C is, on average, 2.6 times larger than observed with AIA. This increase in intensity suggests that AIA is not resolving these events. This result suggests a finely structured dynamic corona embedded in a smoothly varying background.

  18. S/HIC: Robust Identification of Soft and Hard Sweeps Using Machine Learning.

    Directory of Open Access Journals (Sweden)

    Daniel R Schrider

    2016-03-01

    Full Text Available Detecting the targets of adaptive natural selection from whole genome sequencing data is a central problem for population genetics. However, to date most methods have shown sub-optimal performance under realistic demographic scenarios. Moreover, over the past decade there has been a renewed interest in determining the importance of selection from standing variation in adaptation of natural populations, yet very few methods for inferring this model of adaptation at the genome scale have been introduced. Here we introduce a new method, S/HIC, which uses supervised machine learning to precisely infer the location of both hard and soft selective sweeps. We show that S/HIC has unrivaled accuracy for detecting sweeps under demographic histories that are relevant to human populations, and distinguishing sweeps from linked as well as neutrally evolving regions. Moreover, we show that S/HIC is uniquely robust among its competitors to model misspecification. Thus, even if the true demographic model of a population differs catastrophically from that specified by the user, S/HIC still retains impressive discriminatory power. Finally, we apply S/HIC to the case of resequencing data from human chromosome 18 in a European population sample, and demonstrate that we can reliably recover selective sweeps that have been identified earlier using less specific and sensitive methods.

  19. Formin Homology 2 Domain Containing 3 (FHOD3) Variants Associated with Hypertrophic Cardiomyopathy

    Science.gov (United States)

    Wooten, Eric C.; Hebl, Virginia Bartleson; Wolf, Matthew J.; Greytak, Sarah R.; Orr, Nicole; Draper, Isabelle; Calvino, Jenna E.; Kapur, Navin K.; Maron, Martin S.; Kullo, Iftikhar J.; Ommen, Steve R.; Bos, J. Martijn; Ackerman, Michael J.; Huggins, Gordon S.

    2013-01-01

    Background Incomplete penetrance and variable expression of Hypertrophic Cardiomyopathy (HCM) is well appreciated. Common genetic polymorphisms variants that may affect HCM penetrance and expression have been predicted but are not well established. Methods and Results We performed a case-control genome wide association (GWA) study to identify common HCM-associated genetic polymorphisms and then asked whether such common variants were more represented in HCM or could explain the heterogeneity of HCM phenotypes. We identified an intronic FHOD3 variant (rs516514) associated with HCM (OR = 2.45 (95% CI 1.76–3.41), p=1.25 × 10−7) and validated this finding in an independent cohort. Next, we tested FHOD3-V1151I (rs2303510), a non-synonymous variant in partial linkage disequilibrium (LD) with rs516514, and we detected an even stronger association with HCM (p=1.76 × 10−9). While HCM patients were more likely to carry these FHOD3 alleles subjects homozygous for FHOD3-1151I had similar HCM phenotypes as carriers of the V1151 allele. FHOD3 expression is increased in the setting of HCM and both alleles of FHOD3-V1151I were detected in HCM myectomy tissue. Previously FHOD3 was found to be required for formation of the sarcomere and here we demonstrate that its fly homolog fhos is required for normal adult heart systolic contraction. Conclusions Here we demonstrate the association of a common non-synonymous FHOD3 genetic variant with HCM. This discovery further strengthens the potential role of gene mutations and polymorphisms that alter the amino acid sequence of sarcomere proteins and HCM. PMID:23255317

  20. Formin homology 2 domain containing 3 variants associated with hypertrophic cardiomyopathy.

    Science.gov (United States)

    Wooten, Eric C; Hebl, Virginia B; Wolf, Matthew J; Greytak, Sarah R; Orr, Nicole M; Draper, Isabelle; Calvino, Jenna E; Kapur, Navin K; Maron, Martin S; Kullo, Iftikhar J; Ommen, Steve R; Bos, J Martijn; Ackerman, Michael J; Huggins, Gordon S

    2013-02-01

    Incomplete penetrance and variable expression of hypertrophic cardiomyopathy (HCM) is well appreciated. Common genetic polymorphisms variants that may affect HCM penetrance and expression have been predicted but are not well established. We performed a case-control genomewide association study to identify common HCM-associated genetic polymorphisms and then asked whether such common variants were more represented in HCM or could explain the heterogeneity of HCM phenotypes. We identified an intronic FHOD3 variant (rs516514) associated with HCM (odds ratio, 2.45; 95% confidence interval, 1.76-3.41; P=1.25×10(-7)) and validated this finding in an independent cohort. Next, we tested FHOD3-V1151I (rs2303510), a nonsynonymous variant in partial linkage disequilibrium with rs516514, and we detected an even stronger association with HCM (P=1.76×10(-9)). Although HCM patients were more likely to carry these, FHOD3 allele subjects homozygous for FHOD3-1151I had similar HCM phenotypes as carriers of the V1151 allele. FHOD3 expression is increased in the setting of HCM, and both alleles of FHOD3-V1151I were detected in HCM myectomy tissue. Previously, FHOD3 was found to be required for formation of the sarcomere, and here we demonstrate that its fly homolog fhos is required for normal adult heart systolic contraction. Here we demonstrate the association of a common nonsynonymous FHOD3 genetic variant with HCM. This discovery further strengthens the potential role of gene mutations and polymorphisms that alter the amino acid sequence of sarcomere proteins and HCM.

  1. Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

    Science.gov (United States)

    Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

    2016-08-05

    A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. DISP3, a sterol-sensing domain-containing protein that links thyroid hormone action and cholesterol metabolism

    Czech Academy of Sciences Publication Activity Database

    Zíková, Martina; Corlett, Alicia; Bendová, Zdeňka; Pajer, Petr; Bartůněk, Petr

    2009-01-01

    Roč. 23, č. 4 (2009), s. 520-528 ISSN 0888-8809 R&D Projects: GA AV ČR IAA500520705 Grant - others:EC(XE) LSHM-CT-2005-018652 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50110509 Keywords : thyroid hormone receptor * cholesterol metabolism * sterol-sensing domain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.257, year: 2009

  3. Control of genic DNA methylation by a jmjC domain-containing protein in Arabidopsis thaliana.

    Science.gov (United States)

    Saze, Hidetoshi; Shiraishi, Akiko; Miura, Asuka; Kakutani, Tetsuji

    2008-01-25

    Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.

  4. Molecular cloning and functional analysis of nucleotide-binding oligomerization domain-containing protein 1 in rainbow trout, Oncorhynchus mykiss.

    Science.gov (United States)

    Jang, Ju Hye; Kim, Hyun; Kim, Yu Jin; Cho, Ju Hyun

    2016-04-01

    NOD1 has important roles in innate immunity as sensor of microbial components derived from bacterial peptidoglycan. In this study, we identified genes encoding components of the NOD1 signaling pathway, including NOD1 (OmNOD1) and RIP2 (OmRIP2) from rainbow trout, Oncorhynchus mykiss, and investigated whether OmNOD1 has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 treated with NOD1-specific ligand (iE-DAP). The deduced amino acid sequence of OmNOD1 contained conserved CARD, NOD and LRR domains. Loss-of-function and gain-of-function experiments indicated that OmNOD1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmNOD1 in RTH-149 cells treated with iE-DAP decreased the expression of IL-1β, IL-6, IL-8 and TNF-α. Conversely, overexpression of OmNOD1 resulted in up-regulation of IL-1β, IL-6, IL-8 and TNF-α expression. In addition, RIP2 inhibitor (gefitinib) significantly decreased the expression of these pro-inflammatory cytokines induced by iE-DAP in RTH-149 cells. These findings highlight the important role of NOD1 signaling pathway in fish in eliciting innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Sim3C: simulation of Hi-C and Meta3C proximity ligation sequencing technologies.

    Science.gov (United States)

    DeMaere, Matthew Z; Darling, Aaron E

    2017-11-15

    Chromosome conformation capture (3C) and Hi-C DNA sequencing methods have rapidly advanced our understanding of the spatial organization of genomes and metagenomes. Many variants of these protocols have been developed, each with their own strengths. Currently there is no systematic means for simulating sequence data from this family of sequencing protocols, potentially hindering the advancement of algorithms to exploit this new datatype. We describe a computational simulator that, given simple parameters and reference genome sequences, will simulate Hi-C sequencing on those sequences. The simulator models the basic spatial structure in genomes that is commonly observed in Hi-C and 3C datasets, including the distance-decay relationship in proximity ligation, differences in the frequency of interaction within and across chromosomes, and the structure imposed by cells. A means to model the 3D structure of randomly generated topologically associating domains (TADs) is provided. The simulator considers several sources of error common to 3C and Hi-C library preparation and sequencing methods, including spurious proximity ligation events and sequencing error. We have introduced the first comprehensive simulator for 3C and Hi-C sequencing protocols. We expect the simulator to have use in testing of Hi-C data analysis algorithms, as well as more general value for experimental design, where questions such as the required depth of sequencing, enzyme choice, and other decisions can be made in advance in order to ensure adequate statistical power with respect to experimental hypothesis testing. © The Authors 2017. Published by Oxford University Press.

  6. Hi-C Observations of an Active Region Corona, and Investigation of the Underlying Magnetic Structure

    Science.gov (United States)

    Tiwari, S. K.; Alexander, C. E.; Winebarger, A.; Moore, R. L.

    2014-01-01

    The solar corona is much hotter (>=10(exp 6) K) than its surface (approx 6000 K), puzzling astrophysicists for several decades. Active region (AR) corona is again hotter than the quiet Sun (QS) corona by a factor of 4-10. The most widely accepted mechanism that could heat the active region corona is the energy release by current dissipation via reconnection of braided magnetic field structure, first proposed by E. N. Parker three decades ago. The first observational evidence for this mechanism has only recently been presented by Cirtain et al. by using High-resolution Coronal Imager (Hi-C) observations of an AR corona at a spatial resolution of 0.2 arcsec, which is required to resolve the coronal loops, and was not available before the rocket flight of Hi-C in July 2012. The Hi-C project is led by NASA/MSFC. In the case of the QS, work done by convection/granulation on the inter-granular feet of the coronal field lines translates into the heat observed in the corona. In the case of the AR, as here, there could be flux emergence, cancellation/submergence, or shear flows generating large stress and tension in coronal field loops which is released as heat in the corona. We are currently investigating the changes taking place in photospheric feet of the magnetic field involved with brightenings in the Hi-C AR corona. For this purpose, we are also using SDO/AIA data of +/- 2 hours around the 5 minutes Hi-C flight. In the present talk, I will first summarize some of the results of the Hi-C observations and then present some results from our recent analysis on what photospheric processes feed the magnetic energy that dissipates into heat in coronal loops.

  7. Chemokine-like factor-like MARVEL transmembrane domain-containing 3 expression is associated with a favorable prognosis in esophageal squamous cell carcinoma.

    Science.gov (United States)

    Han, Tianci; Shu, Tianci; Dong, Siyuan; Li, Peiwen; Li, Weinan; Liu, Dali; Qi, Ruiqun; Zhang, Shuguang; Zhang, Lin

    2017-05-01

    Decreased expression of human chemokine-like factor-like MARVEL transmembrane domain-containing 3 (CMTM3) has been identified in a number of human tumors and tumor cell lines, including gastric and testicular cancer, and PC3, CAL27 and Tca-83 cell lines. However, the association between CMTM3 expression and the clinicopathological features and prognosis of esophageal squamous cell carcinoma (ESCC) patients remains unclear. The aim of the present study was to investigate the correlation between CMTM3 expression and clinicopathological parameters and prognosis in ESCC. CMTM3 mRNA and protein expression was analyzed in ESCC and paired non-tumor tissues by quantitative real-time polymerase chain reaction, western blotting and immunohistochemical analysis. The Kaplan-Meier method was used to plot survival curves and the Cox proportional hazards regression model was also used for univariate and multivariate survival analysis. The results revealed that CMTM3 mRNA and protein expression levels were lower in 82.5% (30/40) and 75% (30/40) of ESCC tissues, respectively, when compared with matched non-tumor tissues. Statistical analysis demonstrated that CMTM3 expression was significantly correlated with lymph node metastasis (P=0.002) and clinical stage (P<0.001) in ESCC tissues. Furthermore, the survival time of ESCC patients exhibiting low CMTM3 expression was significantly shorter than that of ESCC patients exhibiting high CMTM3 expression (P=0.01). In addition, Kaplan-Meier survival analysis revealed that the overall survival time of patients exhibiting low CMTM3 expression was significantly decreased compared with patients exhibiting high CMTM3 expression (P=0.010). Cox multivariate analysis indicated that CMTM3 protein expression was an independent prognostic predictor for ESCC after resection. This study indicated that CMTM3 expression is significantly decreased in ESCC tissues and CMTM3 protein expression in resected tumors may present an effective prognostic

  8. Disposal demonstration of a high integrity container (HIC) containing an EPICOR-II prefilter from Three Mile Island

    Energy Technology Data Exchange (ETDEWEB)

    McConnell, J.W. Jr.; Tyacke, M.J.; Schmitt, R.C.; Reno, H.W.

    1985-02-01

    A high integrity container (HIC) was developed, tested, and certified for use in disposing of unusual low-level radioactive waste from Three Mile Island Unit 2 (TMI-2). The work was coordinated by EG and G Idaho, Inc. and funded by the US Department of Energy. A disposal demonstration using an HIC containing an EPICOR-II prefilter from TMI-2 was completed at the commercial disposal facility in the State of Washington. A Certification of Compliance was issued by the Department of Social and Health Services of the State of Washington to use the HIC in disposing of up to 50 EPICOR-II prefilters. That Certification of Compliance was issued after rigorous review of the HIC design and test program by the State and by the US Nuclear Regulatory Commission. This report describes the processes of loading, transporting, and disposing of the demonstration HIC and briefly describes the design, testing, and approval effort leading up to the demonstration.

  9. Disposal demonstration of a high integrity container (HIC) containing an EPICOR-II prefilter from Three Mile Island

    International Nuclear Information System (INIS)

    McConnell, J.W. Jr.; Tyacke, M.J.; Schmitt, R.C.; Reno, H.W.

    1985-02-01

    A high integrity container (HIC) was developed, tested, and certified for use in disposing of unusual low-level radioactive waste from Three Mile Island Unit 2 (TMI-2). The work was coordinated by EG and G Idaho, Inc. and funded by the US Department of Energy. A disposal demonstration using an HIC containing an EPICOR-II prefilter from TMI-2 was completed at the commercial disposal facility in the State of Washington. A Certification of Compliance was issued by the Department of Social and Health Services of the State of Washington to use the HIC in disposing of up to 50 EPICOR-II prefilters. That Certification of Compliance was issued after rigorous review of the HIC design and test program by the State and by the US Nuclear Regulatory Commission. This report describes the processes of loading, transporting, and disposing of the demonstration HIC and briefly describes the design, testing, and approval effort leading up to the demonstration

  10. Amino substituted nitrogen heterocycle ureas as kinase insert domain containing receptor (KDR inhibitors: Performance of structure–activity relationship approaches

    Directory of Open Access Journals (Sweden)

    Hayriye Yilmaz

    2015-06-01

    Full Text Available A quantitative structure–activity relationship (QSAR study was performed on a set of amino-substituted nitrogen heterocyclic urea derivatives. Two novel approaches were applied: (1 the simplified molecular input-line entry systems (SMILES based optimal descriptors approach; and (2 the fragment-based simplex representation of molecular structure (SiRMS approach. Comparison with the classic scheme of building up the model and balance of correlation (BC for optimal descriptors approach shows that the BC scheme provides more robust predictions than the classic scheme for the considered pIC50 of the heterocyclic urea derivatives. Comparison of the SMILES-based optimal descriptors and SiRMS approaches has confirmed good performance of both techniques in prediction of kinase insert domain containing receptor (KDR inhibitory activity, expressed as a logarithm of inhibitory concentration (pIC50 of studied compounds.

  11. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Di, E-mail: DiWu@mail.nankai.edu.cn; Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  12. Protein (Cyanobacteria): 428774810 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 65093:254 ... SUA5/yciO/yrdC domain-containing protein Halothece sp. PCC 7418 MSLVSFSELVTGAKEGKVVSFPTDTVPALAVRPDCA...EAIFELKQREATKPLILMGASPEQLWDYVEGTPEEFQVWEQTAQQYFPGQLTLVLPSSSLVRPEVNPKTADTIGIRVPDCAIARQVFASTGVLATTSANRSGQPPLTTPEAINQAFPEVLVLADTEPIVSSGLPSTVAKWTGQDWEILRQGNIYLK

  13. Protein (Cyanobacteria): 505037159 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available ... SUA5/yciO/yrdC domain-containing protein Halothece sp. PCC 7418 MSLVSFSELVTGAKEGKVVSFPTDTVPALAVRPDCAEAIF...ELKQREATKPLILMGASPEQLWDYVEGTPEEFQVWEQTAQQYFPGQLTLVLPSSSLVRPEVNPKTADTIGIRVPDCAIARQVFASTGVLATTSANRSGQPPLTTPEAINQAFPEVLVLADTEPIVSSGLPSTVAKWTGQDWEILRQGNIYLK

  14. Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

    Science.gov (United States)

    Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

    2018-03-15

    Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Three invariant Hi-C interaction patterns: applications to genome assembly.

    Science.gov (United States)

    Oddes, Sivan; Zelig, Aviv; Kaplan, Noam

    2018-04-20

    Assembly of reference-quality genomes from next-generation sequencing data is a key challenge in genomics. Recently, we and others have shown that Hi-C data can be used to address several outstanding challenges in the field of genome assembly. This principle has since been developed in academia and industry, and has been used in the assembly of several major genomes. In this paper, we explore the central principles underlying Hi-C-based assembly approaches, by quantitatively defining and characterizing three invariant Hi-C interaction patterns on which these approaches can build: Intrachromosomal interaction enrichment, distance-dependent interaction decay and local interaction smoothness. Specifically, we evaluate to what degree each invariant pattern holds on a single locus level in different species, cell types and Hi-C map resolutions. We find that these patterns are generally consistent across species and cell types but are affected by sequencing depth, and that matrix balancing improves consistency of loci with all three invariant patterns. Finally, we overview current Hi-C-based assembly approaches in light of these invariant patterns and demonstrate how local interaction smoothness can be used to easily detect scaffolding errors in extremely sparse Hi-C maps. We suggest that simultaneously considering all three invariant patterns may lead to better Hi-C-based genome assembly methods. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. HIC1 loss promotes prostate cancer metastasis by triggering epithelial mesenchymal transition

    Czech Academy of Sciences Publication Activity Database

    Hao, M.; Li, Y.; Wang, J.; Qin, J.; Wang, Y.; Ding, Y.; Jiang, M.; Sun, X.; Zu, L.; Chang, K.; Lin, G.; Du, J.; Kořínek, Vladimír; Ye, D.W.; Wang, J.

    2017-01-01

    Roč. 242, č. 4 (2017), s. 409-420 ISSN 0022-3417 R&D Projects: GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : HIC * prostate cancer * EMT * metastasis Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 6.894, year: 2016

  17. Anti-parallel filament flows and bright dots observed in the EUV with Hi-C

    Science.gov (United States)

    Alexander, C. E.; Regnier, S.; Walsh, R. W.; Winebarger, A. R.; Cirtain, J. W.

    2013-12-01

    The Hi-C instrument imaged the million degree corona at the highest spatial and temporal resolution to date. The instrument imaged a complicated active region which contained several interesting features. Scientists at UCLan in the UK, in collaboration with other members of the Hi-C science team, studied two of these festures: anti-parallel filament flows and bright EUV dots. Plasma flows within prominences/filaments have been observed for many years and hold valuable clues concerning the mass and energy balance within these structures. Evidence of ';counter-steaming' flows has previously been inferred from these cool plasma observations but now, for the first time, these flows have been directly imaged along fundamental filament threads within the million degree corona (at 193 Å). We present observations of an active region filament observed with Hi-C that exhibits anti-parallel flows along adjacent filament threads. The ultra-high spatial and temporal resolution of Hi-C allow the anti-parallel flow velocities to be measured (70 - 80 km/s) and gives an indication of the resolvable thickness of the individual strands (0.8' × 0.1'). The temperature distribution of the plasma flows was estimated to be log T(K) = 5.45 × 0.10 using EM loci analysis. Short-lived, small brightenings sparkling at the edge of the active region, calle EUV Bright Dots (EBDs) were also investigated. EBDs have a characteristic duration of 25 s with a characteristic length of 680 km. These brightenings are not fully resolved by the SDO/AIA instrument at the same wavelength, but can however be identified with respect to the Hi-C location of the EBDs. In addition, EBDs are seen in other chromospheric/coronal channels of SDO/AIA suggesting a temperature between 0.5 and 1.5 MK. Based on a potential field extrapolation from an SDO/HMI magnetogram, the EBDs appear at the footpoints of large-scale trans-equatorial coronal loops. The Hi-C observations provide the first evidence of small-scale EUV

  18. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules RSAD2 CIG5 Radical S-adenosyl methionine domain-containing protein 2 Cytomegalo...virus-induced gene 5 protein, Viperin, Virus inhibitory protein, endoplasmic reticu

  19. Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

    Science.gov (United States)

    Ma, Wenxiu; Ay, Ferhat; Lee, Choli; Gulsoy, Gunhan; Deng, Xinxian; Cook, Savannah; Hesson, Jennifer; Cavanaugh, Christopher; Ware, Carol B; Krumm, Anton; Shendure, Jay; Blau, C Anthony; Disteche, Christine M; Noble, William S; Duan, ZhiJun

    2018-01-31

    The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Reactivation of HIC-1 gene by saRNA inhibits clonogenicity and invasiveness in breast cancer cells.

    Science.gov (United States)

    Zhao, Feng; Pan, Shengli; Gu, Yan; Guo, Shanyu; Dai, Qiancheng; Yu, Yingyan; Zhang, Wei

    2015-01-01

    Hypermethylated in cancer 1 (HIC-1) is a tumor suppressor gene, which is epigenetically silenced in breast cancer. It is known that the loss of HIC-1, caused by promoter hypermethylation, is associated with tumor aggression and poor survival in breast carcinoma. It has been shown that small activating RNA (saRNA) targeting promoter sequences may induce gene re-expression. In the current study, saRNA was used to restore HIC-1 expression, and the effects on colony formation, invasiveness and the cell cycle in breast cancer cells were explored. dsHIC1-2998, an saRNA, exhibited activating efficacy on MCF-7 and MDA-MB-231 cancer cell lines. A clonogenicity assay showed that evident colony inhibition was induced via saRNA-mediated re-expression of HIC-1 in the two cancer cell lines. Reactivation of HIC-1 significantly inhibited cell migration and invasion, resulting in G0/G1 cell cycle arrest in these cell lines. These findings suggest that HIC-1 may be a potential target in gene therapy for the treatment of breast cancer. saRNA may function as a therapeutic option for upregulating tumor suppressor genes in breast cancer.

  1. Comparison of Hi-C results using in-solution versus in-nucleus ligation.

    Science.gov (United States)

    Nagano, Takashi; Várnai, Csilla; Schoenfelder, Stefan; Javierre, Biola-Maria; Wingett, Steven W; Fraser, Peter

    2015-08-26

    Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries. We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

  2. Sparkling extreme-ultraviolet bright dots observed with Hi-C

    International Nuclear Information System (INIS)

    Régnier, S.; Alexander, C. E.; Walsh, R. W.; Winebarger, A. R.; Cirtain, J.; Golub, L.; Korreck, K. E.; Weber, M.; Mitchell, N.; Platt, S.; De Pontieu, B.; Title, A.; Kobayashi, K.; Kuzin, S.; DeForest, C. E.

    2014-01-01

    Observing the Sun at high time and spatial scales is a step toward understanding the finest and fundamental scales of heating events in the solar corona. The high-resolution coronal (Hi-C) instrument has provided the highest spatial and temporal resolution images of the solar corona in the EUV wavelength range to date. Hi-C observed an active region on 2012 July 11 that exhibits several interesting features in the EUV line at 193 Å. One of them is the existence of short, small brightenings 'sparkling' at the edge of the active region; we call these EUV bright dots (EBDs). Individual EBDs have a characteristic duration of 25 s with a characteristic length of 680 km. These brightenings are not fully resolved by the SDO/AIA instrument at the same wavelength; however, they can be identified with respect to the Hi-C location of the EBDs. In addition, EBDs are seen in other chromospheric/coronal channels of SDO/AIA, which suggests a temperature between 0.5 and 1.5 MK. Based on their frequency in the Hi-C time series, we define four different categories of EBDs: single peak, double peak, long duration, and bursty. Based on a potential field extrapolation from an SDO/HMI magnetogram, the EBDs appear at the footpoints of large-scale, trans-equatorial coronal loops. The Hi-C observations provide the first evidence of small-scale EUV heating events at the base of these coronal loops, which have a free magnetic energy of the order of 10 26 erg.

  3. The -271 G>A polymorphism of kinase insert domain-containing receptor gene regulates its transcription level in patients with non-small cell lung cancer

    International Nuclear Information System (INIS)

    An, She-Juan; Chen, Zhi-Hong; Lin, Qiu-Xiong; Su, Jian; Chen, Hua-Jun; Lin, Jia-Ying; Wu, Yi-Long

    2009-01-01

    Kinase insert domain-containing receptor (KDR) plays a critical role in the metastasis of cancer and is used as a molecular target in cancer therapy. We investigated the characteristics of the -271 G>A polymorphism of the KDR gene to gain information that may benefit the development of individualized therapies for patients with non-small cell lung cancer (NSCLC). The -271 G>A polymorphism of the KDR gene in 106 lung cancer patients and 203 healthy control individuals was analyzed by polymerase chain reaction (PCR) and DNA sequencing methods. Real-time quantitative PCR and immunohistochemical methods were used to evaluate KDR mRNA and protein expression levels, respectively, in frozen tumor specimens. The -271 G>A polymorphism was associated with the mRNA expression level of the KDR gene in tumor tissues (t = 2.178, P = 0.032, independent samples t-test). Compared with the AG/GG genotype, the AA genotype was associated with higher KDR mRNA expression in tumor tissues. We found no relationship between the genotype and the KDR protein expression level and no significant difference in the distribution of the KDR gene polymorphism genotypes between lung cancer patients and the control group (χ 2 = 1.269, P = 0.264, Fisher's exact test). This study is the first to show that the -271 G>A polymorphism of the KDR gene may be a functional polymorphism related to the regulation of gene transcription. These findings may have important implications for therapies targeting KDR in patients with NSCLC

  4. Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

    Science.gov (United States)

    Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

    2014-01-01

    Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein

  5. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    Science.gov (United States)

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  6. Tetrazolylhydrazides as Selective Fragment-Like Inhibitors of the JumonjiC-Domain-Containing Histone Demethylase KDM4A.

    Science.gov (United States)

    Rüger, Nicole; Roatsch, Martin; Emmrich, Thomas; Franz, Henriette; Schüle, Roland; Jung, Manfred; Link, Andreas

    2015-11-01

    The JumonjiC-domain-containing histone demethylase 2A (JMJD2A, KDM4A) is a key player in the epigenetic regulation of gene expression. Previous publications have shown that both elevated and lowered enzyme levels are associated with certain types of cancer, and therefore the definite role of KDM4A in oncogenesis remains elusive. To identify a novel molecular starting point with favorable physicochemical properties for the investigation of the physiological role of KDM4A, we screened a number of molecules bearing an iron-chelating moiety by using two independent assays. In this way, we were able to identify 2-(1H-tetrazol-5-yl)acetohydrazide as a novel fragment-like lead structure with low relative molecular mass (Mr =142 Da), low complexity, and an IC50 value of 46.6 μm in a formaldehyde dehydrogenase (FDH)-coupled assay and 2.4 μm in an antibody-based assay. Despite its small size, relative selectivity against two other demethylases could be demonstrated for this compound. This is the first example of a tetrazole group as a warhead in JMJD demethylases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Three-dimensional local ALE-FEM method for fluid flow in domains containing moving boundaries/objects interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Carrington, David Bradley [Los Alamos National Laboratory (LANL), Los Alamos, NM (United States); Monayem, A. K. M. [Univ. of New Mexico, Albuquerque, NM (United States); Mazumder, H. [Univ. of New Mexico, Albuquerque, NM (United States); Heinrich, Juan C. [Univ. of New Mexico, Albuquerque, NM (United States)

    2015-03-05

    A three-dimensional finite element method for the numerical simulations of fluid flow in domains containing moving rigid objects or boundaries is developed. The method falls into the general category of Arbitrary Lagrangian Eulerian methods; it is based on a fixed mesh that is locally adapted in the immediate vicinity of the moving interfaces and reverts to its original shape once the moving interfaces go past the elements. The moving interfaces are defined by separate sets of marker points so that the global mesh is independent of interface movement and the possibility of mesh entanglement is eliminated. The results is a fully robust formulation capable of calculating on domains of complex geometry with moving boundaries or devises that can also have a complex geometry without danger of the mesh becoming unsuitable due to its continuous deformation thus eliminating the need for repeated re-meshing and interpolation. Moreover, the boundary conditions on the interfaces are imposed exactly. This work is intended to support the internal combustion engines simulator KIVA developed at Los Alamos National Laboratories. The model's capabilities are illustrated through application to incompressible flows in different geometrical settings that show the robustness and flexibility of the technique to perform simulations involving moving boundaries in a three-dimensional domain.

  8. Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD 2 in Host Defense during Pneumococcal Pneumonia.

    Directory of Open Access Journals (Sweden)

    Tijmen J Hommes

    Full Text Available Streptococcus (S. pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2-/- and wild-type (Wt alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39, an isogenic capsule locus deletion mutant (D39Δcps or serotype 3 S. pneumoniae (6303 via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.

  9. BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

    Science.gov (United States)

    Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

    2010-11-01

    A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Identification of copy number variations and translocations in cancer cells from Hi-C data.

    Science.gov (United States)

    Chakraborty, Abhijit; Ay, Ferhat

    2017-10-18

    Eukaryotic chromosomes adapt a complex and highly dynamic three-dimensional (3D) structure, which profoundly affects different cellular functions and outcomes including changes in epigenetic landscape and in gene expression. Making the scenario even more complex, cancer cells harbor chromosomal abnormalities (e.g., copy number variations (CNVs) and translocations) altering their genomes both at the sequence level and at the level of 3D organization. High-throughput chromosome conformation capture techniques (e.g., Hi-C), which are originally developed for decoding the 3D structure of the chromatin, provide a great opportunity to simultaneously identify the locations of genomic rearrangements and to investigate the 3D genome organization in cancer cells. Even though Hi-C data has been used for validating known rearrangements, computational methods that can distinguish rearrangement signals from the inherent biases of Hi-C data and from the actual 3D conformation of chromatin, and can precisely detect rearrangement locations de novo have been missing. In this work, we characterize how intra and inter-chromosomal Hi-C contacts are distributed for normal and rearranged chromosomes to devise a new set of algorithms (i) to identify genomic segments that correspond to CNV regions such as amplifications and deletions (HiCnv), (Nurtdinov et al.) to call inter-chromosomal translocations and their boundaries (HiCtrans) from Hi-C experiments, and (iii) to simulate Hi-C data from genomes with desired rearrangements and abnormalities (AveSim) in order to select optimal parameters for and to benchmark the accuracy of our methods. Our results on 10 different cancer cell lines with Hi-C data show that we identify a total number of 105 amplifications and 45 deletions together with 90 translocations, whereas we identify virtually no such events for two karyotypically normal cell lines. Our CNV predictions correlate very well with whole genome sequencing (WGS) data among chromosomes

  11. Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.

    Science.gov (United States)

    Gospodarek, Adrian M; Sun, Weitong; O'Connell, John P; Fernandez, Erik J

    2014-12-05

    In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. HIC1 links retinoic acid signalling to group 3 innate lymphoid cell-dependent regulation of intestinal immunity and homeostasis

    Science.gov (United States)

    Antignano, Frann; Korinek, Vladimir; Underhill, T. Michael

    2018-01-01

    The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity. PMID:29470558

  13. High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zijian; Huang, Shanzhou [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Wang, Huanyu [Department of Thyroid and Breast Surgery, Nanshan District People’s Hospital, Shenzhen, 518000 (China); Wu, Jian [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Chen, Dong [Department of Biliopancreatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Peng, Baogang [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Zhou, Qi, E-mail: hnzhouqi@163.com [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China)

    2016-06-10

    Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. -- Highlights: •HKDC1 is upregulated in HCC. •Patients with high HKDC1 expressions perform worse OS. •Silencing HKDC1 suppresses proliferation and migration. •Silencing HKDC1 represses Wnt/β-catenin signaling pathway.

  14. Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree.

    Directory of Open Access Journals (Sweden)

    Tomoki Yagai

    Full Text Available Cholangiocarcinoma (CC is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC at the peribiliary gland (PBG of large bile ducts and liver stem/progenitor cell (LPC at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5 is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines.SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

  15. Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

    Directory of Open Access Journals (Sweden)

    Si Won Kim

    2017-08-01

    Full Text Available Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2 gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7 cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

  16. Association of kinase insert domain-containing receptor (KDR gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure

    Directory of Open Access Journals (Sweden)

    Shiva Shahsavari

    2015-12-01

    Full Text Available Background: Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR gene are examined that can endanger the life of the fetus in pregnant women. Objective: The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA. Materials and Methods: A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H, and rs2305948 (V297I as well as one tag SNP in the intron region (rs6838752 were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP technique. Haplotype frequency was determined for these three SNPs’ genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. Results: Functional SNP (rs1870377 was highly linked to tag SNP (rs6838752 (D´ value=0. 214; χ2 = 16.44, p<0. 001. K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. Conclusion: In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.

  17. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  18. Commercial disposal of High Integrity Containers (HICs) containing EPICOR-II prefilters from Three Mile Island

    International Nuclear Information System (INIS)

    McConnell, J.W. Jr.; Lynch, R.J.; Tyacke, M.J.

    1985-09-01

    This report describes the processes of loading, transporting, and commercially disposing of 45 High Integrity Containers (HICs), each containing an EPICOR-II prefilter. Also described are the improvements that were applied in the disposition of the 45 commercial EPICOR-II prefilters at the Idaho National Engineering Laboratory (INEL), versus those used for the demonstration unit. The significance of this effort was that the commercial disposal campaign involved the first-of-a-kind production use of a reinforced concrete HIC at the US Ecology, Inc. facility in the State of Washington. This allowed for safe disposal of high-specific-activity ion exchange material in EPICOR-II prefilters generated during the cleanup of the Unit-2 Auxiliary and Fuel Handling Building of the Three Mile Island Nuclear Power Station. 26 figs

  19. HiCRep: assessing the reproducibility of Hi-C data using a stratum-adjusted correlation coefficient.

    Science.gov (United States)

    Yang, Tao; Zhang, Feipeng; Yardımcı, Galip Gürkan; Song, Fan; Hardison, Ross C; Noble, William Stafford; Yue, Feng; Li, Qunhua

    2017-11-01

    Hi-C is a powerful technology for studying genome-wide chromatin interactions. However, current methods for assessing Hi-C data reproducibility can produce misleading results because they ignore spatial features in Hi-C data, such as domain structure and distance dependence. We present HiCRep, a framework for assessing the reproducibility of Hi-C data that systematically accounts for these features. In particular, we introduce a novel similarity measure, the stratum adjusted correlation coefficient (SCC), for quantifying the similarity between Hi-C interaction matrices. Not only does it provide a statistically sound and reliable evaluation of reproducibility, SCC can also be used to quantify differences between Hi-C contact matrices and to determine the optimal sequencing depth for a desired resolution. The measure consistently shows higher accuracy than existing approaches in distinguishing subtle differences in reproducibility and depicting interrelationships of cell lineages. The proposed measure is straightforward to interpret and easy to compute, making it well-suited for providing standardized, interpretable, automatable, and scalable quality control. The freely available R package HiCRep implements our approach. © 2017 Yang et al.; Published by Cold Spring Harbor Laboratory Press.

  20. Generation of two modified mouse alleles of the Hic1 tumor suppressor gene

    Czech Academy of Sciences Publication Activity Database

    Pospíchalová, Vendula; Turečková, Jolana; Fafílek, Bohumil; Vojtěchová, Martina; Krausová, Michaela; Lukáš, Jan; Šloncová, Eva; Takacova, S.; Divoký, V.; Leprince, D.; Plachý, Jiří; Kořínek, Vladimír

    2011-01-01

    Roč. 49, č. 3 (2011), s. 142-151 ISSN 1526-954X R&D Projects: GA ČR(CZ) GA204/07/1567; GA ČR(CZ) GD204/09/H058 Institutional research plan: CEZ:AV0Z50520514 Keywords : Hypermethylated In Cancer 1 * Hic1 tumor suppressor * gene targeting Subject RIV: EB - Gene tics ; Molecular Biology Impact factor: 2.527, year: 2011

  1. Generation of two modified mouse alleles of the Hic1 tumor suppressor gene

    Czech Academy of Sciences Publication Activity Database

    Pospíchalová, Vendula; Turečková, Jolana; Fafílek, Bohumil; Vojtěchová, Martina; Krausová, Michaela; Lukáš, Jan; Šloncová, Eva; Takacova, S.; Divoký, V.; Leprince, D.; Plachý, Jiří; Kořínek, Vladimír

    2011-01-01

    Roč. 49, č. 3 (2011), s. 142-151 ISSN 1526-954X R&D Projects: GA ČR(CZ) GA204/07/1567; GA ČR(CZ) GD204/09/H058 Institutional research plan: CEZ:AV0Z50520514 Keywords : Hypermethylated In Cancer 1 * Hic1 tumor suppressor * gene targeting Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.527, year: 2011

  2. The pipeline fracture behavior and pressure assessment under HIC (Hydrogen induced cracking) environment

    Energy Technology Data Exchange (ETDEWEB)

    Shaohua, Dong [China National Petroleum Corporation (CNPC), Beijing (China); Lianwei, Wang [University of Science and Technology Beijing (USTB), Beijing (China)

    2009-07-01

    As Hydrogen's transmit and diffuse, after gestating for a while, the density of hydrogen around crack tip of pipeline will get to the critical density, and the pipeline material will descend, make critical stress factor, the reason of pipeline Hydrogen Induced Cracking is Hydrogen's transmit and diffuse. The stress factor of Hydrogen Induced Cracking under surroundings-condition of stress is the key that estimate material's rupture behavior. The paper study the relationship among hydrogen concentrate, crack tip stress, stain field, hydrogen diffusion and inner pressure for crack tip process zone, then determined the length of HIC (hydrogen induced cracking) process zone. Based on the theory of propagation which reason micro-crack making core, dislocation model is produced for fracture criteria of HIC, the influence between material and environments under the HIC is analyzed, step by step pipeline maximum load pressure and threshold of J-integrity ( J{sub ISCC} ) is calculated, which is very significant for pipeline safety operation. (author)

  3. High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation.

    Science.gov (United States)

    Nfor, Beckley K; Hylkema, Nienke N; Wiedhaup, Koenraad R; Verhaert, Peter D E M; van der Wielen, Luuk A M; Ottens, Marcel

    2011-12-09

    Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Increased Expression of the NOD-like Receptor Family, Pyrin Domain Containing 3 Inflammasome in Dermatomyositis and Polymyositis is a Potential Contributor to Their Pathogenesis

    Science.gov (United States)

    Yin, Xi; Han, Gen-Cheng; Jiang, Xing-Wei; Shi, Qiang; Pu, Chuan-Qiang

    2016-01-01

    Background: Dermatomyositis (DM) and polymyositis (PM) are common inflammatory myopathies whose immunopathogenic mechanisms remain poorly understood. The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome is a type of cytoplasmic multiprotein inflammasome and is responsible for the activation of inflammatory reactivations. Responding to a wide range of exogenous and endogenous microbial or sterile stimuli, NLRP3 inflammasomes can cleave pro-caspase-1 into active caspase-1, which processes the pro-inflammatory cytokines pro-interleukin (IL)-1β and pro-IL-18 into active and secreted IL-1β and IL-18. The NLRP3 inflammasome is implicated in infectious and sterile inflammatory diseases. However, it remains unclear whether it is involved in the pathogenesis of DM/PM, which we aim to address in our research. Methods: In this study, 22 DM/PM patients and 24 controls were recruited. The protein and RNA expression of IL-1β, IL-18, NLRP3, and caspase-1 in serum and muscle samples were tested and compared between the two groups. Results: The serum IL-1β and IL-18 levels were significantly higher in DM/PM patients than those in the controls by enzyme linked immunosorbent assay (ELISA, DM vs. control, 25.02 ± 8.29 ng/ml vs. 16.49 ± 3.30 ng/ml, P < 0.001; PM vs. control, 26.49 ± 7.79 ng/ml vs. 16.49 ± 3.30 ng/ml, P < 0.001). Moreover, the real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that DM/PM patients exhibited higher RNA expression of IL-1β, IL-18, and NLRP3 in the muscle (for IL-1β, DM vs. control, P = 0.0012, PM vs. control, P = 0.0021; for IL-18, DM vs. control, P = 0.0045, PM vs. control, P = 0.0031; for NLRP3, DM vs. control, P = 0.0017, PM vs. control, P = 0.0006). Moreover, the protein expression of NLRP3 and caspase-1 in muscle samples of DM/PM patients were also significantly elevated compared to that in the muscles of the controls. Conclusions: Our findings demonstrate that the NLRP3

  5. Beyond the Flipped Classroom: A Highly Interactive Cloud-Classroom (HIC) Embedded into Basic Materials Science Courses

    Science.gov (United States)

    Liou, Wei-Kai; Bhagat, Kaushal Kumar; Chang, Chun-Yen

    2016-06-01

    The present study compares the highly interactive cloud-classroom (HIC) system with traditional methods of teaching materials science that utilize crystal structure picture or real crystal structure model, in order to examine its learning effectiveness across three dimensions: knowledge, comprehension and application. The aim of this study was to evaluate the (HIC) system, which incorporates augmented reality, virtual reality and cloud-classroom to teach basic materials science courses. The study followed a pretest-posttest quasi-experimental research design. A total of 92 students (aged 19-20 years), in a second-year undergraduate program, participated in this 18-week-long experiment. The students were divided into an experimental group and a control group. The experimental group (36 males and 10 females) was instructed utilizing the HIC system, while the control group (34 males and 12 females) was led through traditional teaching methods. Pretest, posttest, and delayed posttest scores were evaluated by multivariate analysis of covariance. The results indicated that participants in the experimental group who used the HIC system outperformed the control group, in the both posttest and delayed posttest, across three learning dimensions. Based on these results, the HIC system is recommended to be incorporated in formal materials science learning settings.

  6. Magnetic Structure of Sites of Braiding in Hi-C Active Region

    Science.gov (United States)

    Tiwari, S. K.; Alexander, C. E.; Winebarger, A.; Moore, R. L.

    2014-01-01

    High-resolution Coronal Imager (Hi-C) observations of an active region (AR) corona, at a spatial resolution of 0.2 arcsec, have offered the first direct evidence of field lines braiding, which could deliver sufficient energy to heat the AR corona by current dissipation via magnetic reconnection, a proposal given by Parker three decades ago. The energy required to heat the corona must be transported from the photosphere along the field lines. The mechanism that drives the energy transport to the corona is not yet fully understood. To investigate simultaneous magnetic and intensity structure in and around the AR in detail, we use SDO/HMI+AIA data of + / - 2 hours around the 5 minute Hi-C flight. In the case of the QS, work done by convection/granulation on the inter-granular feet of the coronal field lines probably translates into the heat observed in the corona. In the case of the AR, as here, there could be flux emergence, cancellation/submergence, or shear flows generating large stress and tension in coronal field loops which is released as heat in the corona. However, to the best of our knowledge, there is no observational evidence available to these processes. We investigate the changes taking place in the photospheric feet of the magnetic field involved with brightenings in the Hi-C AR corona. Using HMI 45s magnetograms of four hours we find that, out of the two Hi-C sub-regions where the braiding of field lines were recently detected, flux emergence takes place in one region and flux cancellation in the other. The field in these sub-regions are highly sheared and have apparent high speed plasma flows at their feet. Therefore, shearing flows plausibly power much of the coronal and transition region heating in these areas of the AR. In addition, the presence of large flux emergence/cancellation strongly suggests that the work done by these processes on the pre-existing field also drives much of the observed heating.

  7. Microstructure and properties of low manganese and niobium containing HIC pipeline steel

    International Nuclear Information System (INIS)

    Nayak, S.S.; Misra, R.D.K.; Hartmann, J.; Siciliano, F.; Gray, J.M.

    2008-01-01

    The paper describes the concept of using low manganese content in pipeline steels for hydrogen-induced cracking (HIC) applications. The microstructure of thermomechanically processed pipeline steel primarily consisted of polygonal ferrite and low fraction of pearlite. The cleanliness of the steel was evident as was the absence of centerline segregation. The microstructure contained high dislocation density, sub-boundaries and dislocation substructures. Fine-scale precipitation of niobium carbides occurred on parallel array of dislocations and on random dislocations that followed [0 0 1] NbC //[0 0 1] α-Fe relationship with the ferrite matrix

  8. Effects of resveratrol, grape juice or red wine consumption Irisin levels and fibronectin type III domain containing protein 5 and uncoupoling protein gene expression modulation in rats

    Directory of Open Access Journals (Sweden)

    Gabrielle de Souza Rocha

    2016-02-01

    Conclusion: Resveratrol and grape juice were able to increase muscle tissue FNDC5 gene expression, and high-fat diet, red wine and resveratrol, increased UCP2 gene expression in this tissue. Grape juice was capable of increasing adipose tissue UCP2 gene expression. High-fat diet, isolated or associated to beverages rich in polyphenols, have decreased FNDC5 gene expression in adipose tissue. Nevertheless, the interventions did not affect irisin levels.

  9. Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.

    Science.gov (United States)

    Nagano, Takashi; Lubling, Yaniv; Yaffe, Eitan; Wingett, Steven W; Dean, Wendy; Tanay, Amos; Fraser, Peter

    2015-12-01

    Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

  10. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

    DEFF Research Database (Denmark)

    Pandey, A.; Blagoev, B.; Kratchmarova, I.

    2002-01-01

    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling mo...

  11. Commercial disposal of high integrity containers (HICs) containing EPICOR-II prefilters from Three Mile Island: Reflections and projections

    International Nuclear Information System (INIS)

    McConnell, J.W. Jr.; Schmitt, R.C.

    1986-09-01

    The processes of loading, transporting, and commerically disposing of 46 EPICOR-II prefilters, each contained in a High Integrity Container (HIC), are described. Also described are participation of the regulatory agencies and industrial organizations in combining their efforts to accomplish this task. The significant aspect of the task was that the commerical disposal involved the first-of-a-kind production use of a reinforced concrete HIC at the US Ecology, Inc., facility in the State of Washington. The same type of container probably can be used in below- or above-ground disposal of other types of high specific activity, low-level nuclear wastes. 14 refs., 4 figs

  12. Juicebox.js Provides a Cloud-Based Visualization System for Hi-C Data.

    Science.gov (United States)

    Robinson, James T; Turner, Douglass; Durand, Neva C; Thorvaldsdóttir, Helga; Mesirov, Jill P; Aiden, Erez Lieberman

    2018-02-28

    Contact mapping experiments such as Hi-C explore how genomes fold in 3D. Here, we introduce Juicebox.js, a cloud-based web application for exploring the resulting datasets. Like the original Juicebox application, Juicebox.js allows users to zoom in and out of such datasets using an interface similar to Google Earth. Juicebox.js also has many features designed to facilitate data reproducibility and sharing. Furthermore, Juicebox.js encodes the exact state of the browser in a shareable URL. Creating a public browser for a new Hi-C dataset does not require coding and can be accomplished in under a minute. The web app also makes it possible to create interactive figures online that can complement or replace ordinary journal figures. When combined with Juicer, this makes the entire process of data analysis transparent, insofar as every step from raw reads to published figure is publicly available as open source code. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Prediction of protein retention times in hydrophobic interaction chromatography by robust statistical characterization of their atomic-level surface properties.

    NARCIS (Netherlands)

    Hanke, A.T.; Klijn, M.E.; Verhaert, P.D.; Wielen, van der L.; Ottens, M.; Eppink, M.H.M.; Sandt, van de E.J.A.X.

    2016-01-01

    The correlation between the dimensionless retention times (DRT) of proteins in hydrophobic interaction chromatography (HIC) and their surface properties were investigated. A ternary atomic-level hydrophobicity scale was used to calculate the distribution of local average hydrophobicity across the

  14. Characterization of a novel cell wall binding domain-containing Staphylococcus aureus endolysin LysSA97.

    Science.gov (United States)

    Chang, Yoonjee; Ryu, Sangryeol

    2017-01-01

    Endolysin from Staphylococcus aureus phage SA97 (LysSA97) was cloned and investigated. LysSA97 specifically lyse the staphylococcal strains and effectively disrupted staphylococcal biofilms. Bioinformatic analysis of LysSA97 revealed a novel putative cell wall binding domain (CBD) as well as two enzymatically active domains (EADs) containing cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, PF05257) and N-acetylmuramoyl-L-alanine amidase (Amidase-3, PF01520) domains. Comparison of 98 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61 % of the staphylococcal endolysins have a src-homology 3 (SH3, PF08460) domain as CBD, but the remaining 19.39 %, including LysSA97, has a putative C-terminal CBD with no homology to the known CBD. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 showed a specific binding spectrum against staphylococcal cells comparable to SH3 domain (PF08460), suggesting that the C-terminal domain of LysSA97 is a novel CBD of staphylococcal endolysins.

  15. HiC-spector: a matrix library for spectral and reproducibility analysis of Hi-C contact maps.

    Science.gov (United States)

    Yan, Koon-Kiu; Yardimci, Galip Gürkan; Yan, Chengfei; Noble, William S; Gerstein, Mark

    2017-07-15

    Genome-wide proximity ligation based assays like Hi-C have opened a window to the 3D organization of the genome. In so doing, they present data structures that are different from conventional 1D signal tracks. To exploit the 2D nature of Hi-C contact maps, matrix techniques like spectral analysis are particularly useful. Here, we present HiC-spector, a collection of matrix-related functions for analyzing Hi-C contact maps. In particular, we introduce a novel reproducibility metric for quantifying the similarity between contact maps based on spectral decomposition. The metric successfully separates contact maps mapped from Hi-C data coming from biological replicates, pseudo-replicates and different cell types. Source code in Julia and Python, and detailed documentation is available at https://github.com/gersteinlab/HiC-spector . koonkiu.yan@gmail.com or mark@gersteinlab.org. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  16. Promoter-enhancer interactions identified from Hi-C data using probabilistic models and hierarchical topological domains.

    Science.gov (United States)

    Ron, Gil; Globerson, Yuval; Moran, Dror; Kaplan, Tommy

    2017-12-21

    Proximity-ligation methods such as Hi-C allow us to map physical DNA-DNA interactions along the genome, and reveal its organization into topologically associating domains (TADs). As the Hi-C data accumulate, computational methods were developed for identifying domain borders in multiple cell types and organisms. Here, we present PSYCHIC, a computational approach for analyzing Hi-C data and identifying promoter-enhancer interactions. We use a unified probabilistic model to segment the genome into domains, which we then merge hierarchically and fit using a local background model, allowing us to identify over-represented DNA-DNA interactions across the genome. By analyzing the published Hi-C data sets in human and mouse, we identify hundreds of thousands of putative enhancers and their target genes, and compile an extensive genome-wide catalog of gene regulation in human and mouse. As we show, our predictions are highly enriched for ChIP-seq and DNA accessibility data, evolutionary conservation, eQTLs and other DNA-DNA interaction data.

  17. Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

    Science.gov (United States)

    Lappas, Martha

    2016-04-01

    Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1β and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1β, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1β-induced IκB-α protein degradation and NF-κB DNA binding activity. The

  18. JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis.

    Science.gov (United States)

    He, Zhimin; Wu, Junyu; Su, Xiaonan; Zhang, Ye; Pan, Lixia; Wei, Huimin; Fang, Qiang; Li, Haitao; Wang, Da-Liang; Sun, Fang-Lin

    2016-02-26

    Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Hi-Corrector: a fast, scalable and memory-efficient package for normalizing large-scale Hi-C data.

    Science.gov (United States)

    Li, Wenyuan; Gong, Ke; Li, Qingjiao; Alber, Frank; Zhou, Xianghong Jasmine

    2015-03-15

    Genome-wide proximity ligation assays, e.g. Hi-C and its variant TCC, have recently become important tools to study spatial genome organization. Removing biases from chromatin contact matrices generated by such techniques is a critical preprocessing step of subsequent analyses. The continuing decline of sequencing costs has led to an ever-improving resolution of the Hi-C data, resulting in very large matrices of chromatin contacts. Such large-size matrices, however, pose a great challenge on the memory usage and speed of its normalization. Therefore, there is an urgent need for fast and memory-efficient methods for normalization of Hi-C data. We developed Hi-Corrector, an easy-to-use, open source implementation of the Hi-C data normalization algorithm. Its salient features are (i) scalability-the software is capable of normalizing Hi-C data of any size in reasonable times; (ii) memory efficiency-the sequential version can run on any single computer with very limited memory, no matter how little; (iii) fast speed-the parallel version can run very fast on multiple computing nodes with limited local memory. The sequential version is implemented in ANSI C and can be easily compiled on any system; the parallel version is implemented in ANSI C with the MPI library (a standardized and portable parallel environment designed for solving large-scale scientific problems). The package is freely available at http://zhoulab.usc.edu/Hi-Corrector/. © The Author 2014. Published by Oxford University Press.

  20. ClusterTAD: an unsupervised machine learning approach to detecting topologically associated domains of chromosomes from Hi-C data.

    Science.gov (United States)

    Oluwadare, Oluwatosin; Cheng, Jianlin

    2017-11-14

    With the development of chromosomal conformation capturing techniques, particularly, the Hi-C technique, the study of the spatial conformation of a genome is becoming an important topic in bioinformatics and computational biology. The Hi-C technique can generate genome-wide chromosomal interaction (contact) data, which can be used to investigate the higher-level organization of chromosomes, such as Topologically Associated Domains (TAD), i.e., locally packed chromosome regions bounded together by intra chromosomal contacts. The identification of the TADs for a genome is useful for studying gene regulation, genomic interaction, and genome function. Here, we formulate the TAD identification problem as an unsupervised machine learning (clustering) problem, and develop a new TAD identification method called ClusterTAD. We introduce a novel method to represent chromosomal contacts as features to be used by the clustering algorithm. Our results show that ClusterTAD can accurately predict the TADs on a simulated Hi-C data. Our method is also largely complementary and consistent with existing methods on the real Hi-C datasets of two mouse cells. The validation with the chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq) data shows that the domain boundaries identified by ClusterTAD have a high enrichment of CTCF binding sites, promoter-related marks, and enhancer-related histone modifications. As ClusterTAD is based on a proven clustering approach, it opens a new avenue to apply a large array of clustering methods developed in the machine learning field to the TAD identification problem. The source code, the results, and the TADs generated for the simulated and real Hi-C datasets are available here: https://github.com/BDM-Lab/ClusterTAD .

  1. Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family.

    Science.gov (United States)

    Lesne, Elodie; Dupré, Elian; Lensink, Marc F; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2018-02-27

    switches to phosphatase activity in response to modulating signals. Understanding the molecular regulation of those proteins might lead to new antibacterial strategies. Here, we show that the linker regions between the perception and the enzymatic domains shift between distinct states of conformation in an alternating manner in response to signals and that their antagonistic changes control sensor kinase activity. These linker regions and mechanistic principles appear to be conserved among BvgS homologues, irrespective of the presence or absence of an intervening domain between the perception and the enzymatic domains. This work has thus uncovered general molecular mechanisms that regulate activity of sensor kinases in the BvgS family. Copyright © 2018 Lesne et al.

  2. Nucleotide-binding oligomerization domain containing 1 (NOD1 haplotypes and single nucleotide polymorphisms modify susceptibility to inflammatory bowel diseases in a New Zealand caucasian population: a case-control study

    Directory of Open Access Journals (Sweden)

    Barclay Murray L

    2009-03-01

    Full Text Available Abstract Background The nucleotide-binding oligomerization domain containing 1 (NOD1 gene encodes a pattern recognition receptor that senses pathogens, leading to downstream responses characteristic of innate immunity. We investigated the role of NOD1 single nucleotide polymorphisms (SNPs on IBD risk in a New Zealand Caucasian population, and studied Nod1 expression in response to bacterial invasion in the Caco2 cell line. Findings DNA samples from 388 Crohn's disease (CD, 405 ulcerative colitis (UC, 27 indeterminate colitis patients and 201 randomly selected controls, from Canterbury, New Zealand were screened for 3 common SNPs in NOD1, using the MassARRAY® iPLEX Gold assay. Transcriptional activation of the protein produced by NOD1 (Nod1 was studied after infection of Caco2 cells with Escherichia coli LF82. Carrying the rs2075818 G allele decreased the risk of CD (OR = 0.66, 95% CI = 0.50–0.88, p Conclusion The NOD1 gene is important in signalling invasion of colonic cells by pathogenic bacteria, indicative of its' key role in innate immunity. Carrying specific SNPs in this gene significantly modifies the risk of CD and/or UC in a New Zealand Caucasian population.

  3. GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Bohmdorfer, G.; Schleiffer, A.; Brunmeir, R.; Ferscha, S.; Nizhynska, V.; Kozák, Jaroslav; Angelis, Karel; Kreil, D. P.; Schweizer, D.

    2011-01-01

    Roč. 67, č. 3 (2011), s. 420-433 ISSN 0960-7412 R&D Projects: GA MŠk 1M0505; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50380511 Keywords : structural maintenance of chromosomes * DNA repair * somatic homologous recombination Subject RIV: EI - Biotechnology ; Bionics Impact factor: 6.160, year: 2011

  4. Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

    Science.gov (United States)

    Brahma, Biswajit; Patra, Mahesh Chandra; Mishra, Purusottam; De, Bidhan Chandra; Kumar, Sushil; Maharana, Jitendra; Vats, Ashutosh; Ahlawat, Sonika; Datta, Tirtha Kumar; De, Sachinandan

    2016-04-01

    Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

    DEFF Research Database (Denmark)

    Becker-Heck, Anita; Zohn, Irene E; Okabe, Noriko

    2011-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right org...

  6. Inhibition of Fas-associated death domain-containing protein (FADD protects against myocardial ischemia/reperfusion injury in a heart failure mouse model.

    Directory of Open Access Journals (Sweden)

    Qian Fan

    Full Text Available As technological interventions treating acute myocardial infarction (MI improve, post-ischemic heart failure increasingly threatens patient health. The aim of the current study was to test whether FADD could be a potential target of gene therapy in the treatment of heart failure.Cardiomyocyte-specific FADD knockout mice along with non-transgenic littermates (NLC were subjected to 30 minutes myocardial ischemia followed by 7 days of reperfusion or 6 weeks of permanent myocardial ischemia via the ligation of left main descending coronary artery. Cardiac function were evaluated by echocardiography and left ventricular (LV catheterization and cardiomyocyte death was measured by Evans blue-TTC staining, TUNEL staining, and caspase-3, -8, and -9 activities. In vitro, H9C2 cells transfected with ether scramble siRNA or FADD siRNA were stressed with chelerythrin for 30 min and cleaved caspase-3 was assessed.FADD expression was significantly decreased in FADD knockout mice compared to NLC. Ischemia/reperfusion (I/R upregulated FADD expression in NLC mice, but not in FADD knockout mice at the early time. FADD deletion significantly attenuated I/R-induced cardiac dysfunction, decreased myocardial necrosis, and inhibited cardiomyocyte apoptosis. Furthermore, in 6 weeks long term permanent ischemia model, FADD deletion significantly reduced the infarct size (from 41.20 ± 3.90% in NLC to 26.83 ± 4.17% in FADD deletion, attenuated myocardial remodeling, improved cardiac function and improved survival. In vitro, FADD knockdown significantly reduced chelerythrin-induced the level of cleaved caspase-3.Taken together, our results suggest FADD plays a critical role in post-ischemic heart failure. Inhibition of FADD retards heart failure progression. Our data supports the further investigation of FADD as a potential target for genetic manipulation in the treatment of heart failure.

  7. GOTHiC, a probabilistic model to resolve complex biases and to identify real interactions in Hi-C data.

    Directory of Open Access Journals (Sweden)

    Borbala Mifsud

    Full Text Available Hi-C is one of the main methods for investigating spatial co-localisation of DNA in the nucleus. However, the raw sequencing data obtained from Hi-C experiments suffer from large biases and spurious contacts, making it difficult to identify true interactions. Existing methods use complex models to account for biases and do not provide a significance threshold for detecting interactions. Here we introduce a simple binomial probabilistic model that resolves complex biases and distinguishes between true and false interactions. The model corrects biases of known and unknown origin and yields a p-value for each interaction, providing a reliable threshold based on significance. We demonstrate this experimentally by testing the method against a random ligation dataset. Our method outperforms previous methods and provides a statistical framework for further data analysis, such as comparisons of Hi-C interactions between different conditions. GOTHiC is available as a BioConductor package (http://www.bioconductor.org/packages/release/bioc/html/GOTHiC.html.

  8. GOTHiC, a probabilistic model to resolve complex biases and to identify real interactions in Hi-C data.

    Science.gov (United States)

    Mifsud, Borbala; Martincorena, Inigo; Darbo, Elodie; Sugar, Robert; Schoenfelder, Stefan; Fraser, Peter; Luscombe, Nicholas M

    2017-01-01

    Hi-C is one of the main methods for investigating spatial co-localisation of DNA in the nucleus. However, the raw sequencing data obtained from Hi-C experiments suffer from large biases and spurious contacts, making it difficult to identify true interactions. Existing methods use complex models to account for biases and do not provide a significance threshold for detecting interactions. Here we introduce a simple binomial probabilistic model that resolves complex biases and distinguishes between true and false interactions. The model corrects biases of known and unknown origin and yields a p-value for each interaction, providing a reliable threshold based on significance. We demonstrate this experimentally by testing the method against a random ligation dataset. Our method outperforms previous methods and provides a statistical framework for further data analysis, such as comparisons of Hi-C interactions between different conditions. GOTHiC is available as a BioConductor package (http://www.bioconductor.org/packages/release/bioc/html/GOTHiC.html).

  9. HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

    Science.gov (United States)

    Paget, Sonia; Dubuissez, Marion; Dehennaut, Vanessa; Nassour, Joe; Harmon, Brennan T; Spruyt, Nathalie; Loison, Ingrid; Abbadie, Corinne; Rood, Brian R; Leprince, Dominique

    2017-01-10

    The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFβR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

  10. The NmrA-like family domain containing 1 pseudogene Loc344887 is amplified in gallbladder cancer and promotes epithelial-mesenchymal transition.

    Science.gov (United States)

    Wu, Xiao-Cai; Wang, Shou-Hua; Ou, Hong-Hui; Zhu, Bing; Zhu, Yong; Zhang, Qi; Yang, Yang; Li, Hua

    2017-09-01

    The expression pattern and biological role of long non-coding RNA (lncRNA) in cancer has been reported to be involved in many cancers. Here, we report the expression and biological role of a newly discovered lncRNA NmrA-like family domain containing 1 pseudogene (Loc344887) in gallbladder cancer (GBC). In this study, we found that the expression of Loc344887 was significantly elevated in GBC tissues and cell lines when compared with matched normal tissues and normal epithelial bile duct cell line, respectively. High Loc344887 was associated with larger tumor size. Loc344887 was upregulated significantly after ectopic expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in GBC cells. Downregulation of Loc344887 in GBC cells suppressed cell proliferation, blocked cells in G0/S phase, and decreased the migration and invasion cell numbers. In addition, downregulation of Loc344887 decreased the expression of transcription factor Twist, mesenchymal marker Vimentin, and N-cadherin and increased the expression of epithelial maker E-cadherin, which could prompt a mesenchymal-to-epithelial transition phenotype. These results demonstrated that Loc344887 might contribute to cell proliferation and epithelial-to-mesenchymal transition process in GBC, which might be a potential therapeutic target. © 2017 John Wiley & Sons A/S.

  11. CKLF-Like MARVEL Transmembrane Domain-Containing Member 3 (CMTM3) Inhibits the Proliferation and Tumorigenisis in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Li, Wujun; Zhang, Shaobo

    2017-01-26

    The CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), a member of the CMTM family, was found in several human tumors and plays an important role in the development and progression of tumors. However, the role of CMTM3 in hepatocellular carcinoma (HCC) remains largely unknown. Thus, in the present study, we explored its expression pattern in human HCC cell lines, as well as its functions in HCC cells. Our results demonstrated that the expression of CMTM3 is lowly expressed in HCC cell lines. In vitro, we found that overexpression of CMTM3 obviously inhibited the proliferation, invasion, and EMT process in HCC cells. Furthermore, overexpression of CMTM3 significantly downregulated the expression levels of phosphorylation of JAK2 and STAT3 in HepG2 cells. In vivo, overexpression of CMTM3 attenuated the tumor growth in Balb/c nude mice. In conclusion, we demonstrated that CMTM3 could play an important role in HCC metastasis by EMT induction via, at least partially, suppressing the JAK2/STAT3 signaling pathway. Therefore, CMTM3 may serve as a potential molecular target in the prevention and/or treatment of HCC invasion and metastasis.

  12. Competitive interaction of agonists and antagonists with 5-HT3 recognition sites in membranes of neuroblastoma cells labelled with [3H]ICS 205-930

    International Nuclear Information System (INIS)

    Hoyer, D.; Neijt, H.C.; Karpf, A.

    1989-01-01

    [3H]ICS 205-930 labelled 5-HT3 recognition sites in membranes prepared from murine neuroblastoma N1E-115 cells. Binding was rapid, reversible, saturable and stereoselective to an apparently homogeneous population of sites. Kinetic studies revealed that agonists and antagonists produced a monophasic dissociation reaction of [3H]ICS 205-930 from its recognition sites. The dissociation rate constant of the radioligand was similar whether the dissociation was induced by an agonist or an antagonist. Competition studies carried out with agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. Competition curves were best fit for a 1 site model. [3H]ICS 205-930 binding sites displayed the pharmacological profile of a 5-HT3 receptor. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were apparently competitive in nature, as demonstrated in kinetic and equilibrium experiments. In saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabelled agonists and antagonists, apparent Bmax values were not reduced whereas apparent Kd values were increased in the presence of competing ligands. There was a good agreement between apparent pKB values calculated for the competing ligands in saturation experiments and pKd values calculated from competition experiments. The present data demonstrate that [3H]ICS 205-930 labels a homogeneous population of sites at which agonists and antagonists interact competitively

  13. Protein: MPB2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPB2 Ubiquitin ligases WWP1 WWP1 NEDD4-like E3 ubiquitin-protein ligase WWP1 Atrophin-1-interacting pr...otein 5, WW domain-containing protein 1 9606 Homo sapiens Q9H0M0 11059 2OP7, 1ND7 11059 ...

  14. Unique Features of the Anti-parallel, Heterodimeric Coiled-coil Interaction between Methyl-cytosine Binding Domain 2 (MBD2) Homologues and GATA Zinc Finger Domain Containing 2A (GATAD2A/p66α)*

    Science.gov (United States)

    Walavalkar, Ninad M.; Gordon, Nathaniel; Williams, David C.

    2013-01-01

    The methyl-cytosine binding domain 2 (MBD2)-nucleosome remodeling and deacetylase (NuRD) complex recognizes methylated DNA and silences expression of associated genes through histone deacetylase and nucleosome remodeling functions. Our previous structural work demonstrated that a coiled-coil interaction between MBD2 and GATA zinc finger domain containing 2A (GATAD2A/p66α) proteins recruits the chromodomain helicase DNA-binding protein (CHD4/Mi2β) to the NuRD complex and is necessary for MBD2-mediated DNA methylation-dependent gene silencing in vivo (Gnanapragasam, M. N., Scarsdale, J. N., Amaya, M. L., Webb, H. D., Desai, M. A., Walavalkar, N. M., Wang, S. Z., Zu Zhu, S., Ginder, G. D., and Williams, D. C., Jr. (2011) p66α-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex. Proc. Natl. Acad. Sci. U.S.A. 108, 7487–7492). The p66α-MBD2 interaction differs from most coiled-coils studied to date by forming an anti-parallel heterodimeric complex between two peptides that are largely monomeric in isolation. To further characterize unique features of this complex that drive heterodimeric specificity and high affinity binding, we carried out biophysical analyses of MBD2 and the related homologues MBD3, MBD3-like protein 1 (MBD3L1), and MBD3-like protein 2 (MBD3L2) as well as specific mutations that modify charge-charge interactions and helical propensity of the coiled-coil domains. Analytical ultracentrifugation analyses show that the individual peptides remain monomeric in isolation even at 300 μm in concentration for MBD2. Circular dichroism analyses demonstrate a direct correlation between helical content of the coiled-coil domains in isolation and binding affinity for p66α. Furthermore, complementary electrostatic surface potentials and inherent helical content of each peptide are necessary to maintain high-affinity association. These factors lead to a binding affinity hierarchy of p66α for the

  15. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

    International Nuclear Information System (INIS)

    Gorgani-Firuzjaee, Sattar; Adeli, Khosrow; Meshkani, Reza

    2015-01-01

    The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway

  16. Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gorgani-Firuzjaee, Sattar [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of); Adeli, Khosrow [Division of Clinical Biochemistry, The Hospital for Sick Children, University of Toronto, Toronto (Canada); Meshkani, Reza, E-mail: rmeshkani@tums.ac.ir [Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran (Iran, Islamic Republic of)

    2015-08-21

    The serine–threonine kinase Akt regulates proliferation and survival by phosphorylating a network of protein substrates; however, the role of a negative regulator of the Akt pathway, the SH2-domain-containing inositol 5-phosphatase (SHIP2) in apoptosis of the hepatocytes, remains unknown. In the present study, we studied the molecular mechanisms linking SHIP2 expression to apoptosis using overexpression or suppression of SHIP2 gene in HepG2 cells exposed to palmitate (0.5 mM). Overexpression of the dominant negative mutant SHIP2 (SHIP2-DN) significantly reduced palmitate-induced apoptosis in HepG2 cells, as these cells had increased cell viability, decreased apoptotic cell death and reduced the activity of caspase-3, cytochrome c and poly (ADP-ribose) polymerase. Overexpression of the wild-type SHIP2 gene led to a massive apoptosis in HepG2 cells. The protection from palmitate-induced apoptosis by SHIP2 inhibition was accompanied by a decrease in the generation of reactive oxygen species (ROS). In addition, SHIP2 inhibition was accompanied by an increased Akt and FOXO-1 phosphorylation, whereas overexpression of the wild-type SHIP2 gene had the opposite effects. Taken together, these findings suggest that SHIP2 expression level is an important determinant of hepatic lipoapotosis and its inhibition can potentially be a target in treatment of hepatic lipoapoptosis in diabetic patients. - Highlights: • Lipoapoptosis is the major contributor to the development of NAFLD. • The PI3-K/Akt pathway regulates apoptosis in different cells. • The role of negative regulator of this pathway, SHIP2 in lipoapoptosis is unknown. • SHIP2 inhibition significantly reduces palmitate-induced apoptosis in HepG2 cells. • SHIP2 inhibition prevents palmitate induced-apoptosis by regulating Akt/FOXO1 pathway.

  17. Association of T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3 polymorphisms with susceptibility and disease progression of HBV infection.

    Directory of Open Access Journals (Sweden)

    Jingyu Liao

    Full Text Available PURPOSE: T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3 plays an important role in regulating T cells in hepatitis B virus (HBV infection and hepatocellular carcinoma (HCC. However, few researches have reported the association of Tim-3 genetic variants with susceptibility and progression of HBV infection. In this study, we focused on the association of Tim-3 polymorphisms with HBV infection, HBsAg seroclearance and hepatocellular carcinoma. METHODS: A total of 800 subjects were involved in this study. Four groups were studied here, including HBV, HBsAg seroclearance, HBV-associated HCC and healthy controls. Three single-nucleotide polymorphisms (SNPs of Tim-3, rs246871, rs25855 and rs31223 were genotyped to analyze the association of Tim-3 polymorphisms with susceptibility and disease progression of HBV infection. RESULTS: Our study found that rs31223 and rs246871 were associated with disease progression of HBV infection, while none of the three SNPs was relevant to HBV susceptibility. The minor allele "C" of rs31223 was found to be associated with an increased probability of HBsAg seroclearance (P = 0.033 and genotype "CC" of rs246871 to be associated with an increased probability of HBV-associated HCC (P = 0.007. In accordance, haplotypic analysis of the three polymorphisms also showed that the haplotype block CGC* and TGC* were significantly associated with HBsAg seroclearance (P<0.05 while haplotype block CAT*, CGT*, TAC* and TGT* were significantly associated with HBV-associated HCC (all P<0.05. CONCLUSIONS: Genetic variants of Tim-3 have an important impact on disease progression of HBV infection. With specific Tim-3 polymorphisms, patients infected with HBV could be potential candidates of HCC and HBsAg seroclearance.

  18. Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

    Science.gov (United States)

    Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

    2015-05-29

    Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Single-cell Hi-C bridges microscopy and genome-wide sequencing approaches to study 3D chromatin organization.

    Science.gov (United States)

    Ulianov, Sergey V; Tachibana-Konwalski, Kikue; Razin, Sergey V

    2017-10-01

    Recent years have witnessed an explosion of the single-cell biochemical toolbox including chromosome conformation capture (3C)-based methods that provide novel insights into chromatin spatial organization in individual cells. The observations made with these techniques revealed that topologically associating domains emerge from cell population averages and do not exist as static structures in individual cells. Stochastic nature of the genome folding is likely to be biologically relevant and may reflect the ability of chromatin fibers to adopt a number of alternative configurations, some of which could be transiently stabilized and serve regulatory purposes. Single-cell Hi-C approaches provide an opportunity to analyze chromatin folding in rare cell types such as stem cells, tumor progenitors, oocytes, and totipotent cells, contributing to a deeper understanding of basic mechanisms in development and disease. Here, we review key findings of single-cell Hi-C and discuss possible biological reasons and consequences of the inferred dynamic chromatin spatial organization. © 2017 WILEY Periodicals, Inc.

  20. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules Zc3h12a Mcpip, Mcpip1 Ribonuclease ZC3H12A MCP-induced pro...tein 1, Zinc finger CCCH domain-containing protein 12A 10090 Mus musculus 230738 Q5D1E7 Q5D1E7 19322177 ...

  1. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules Rsad2 Vig1 Radical S-adenosyl methionine domain-containing pr...otein 2 Viperin, Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible 10090 Mus musculus 58185 Q8CBB9 21435586 ...

  2. Magic Numbers in Protein Structures

    DEFF Research Database (Denmark)

    Lindgård, Per-Anker; Bohr, Henrik

    1996-01-01

    A homology measure for protein fold classes has been constructed by locally projecting consecutive secondary structures onto a lattice. Taking into account hydrophobic forces we have found a mechanism for formation of domains containing magic numbers of secondary structures and multipla of these ......A homology measure for protein fold classes has been constructed by locally projecting consecutive secondary structures onto a lattice. Taking into account hydrophobic forces we have found a mechanism for formation of domains containing magic numbers of secondary structures and multipla...

  3. Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols.

    Science.gov (United States)

    Lando, David; Stevens, Tim J; Basu, Srinjan; Laue, Ernest D

    2018-01-01

    Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy. We then carry out a systematic evaluation of recently published single-cell Hi-C datasets to establish a computational approach for the evaluation of single-cell Hi-C protocols. We show that the calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range cis- and trans-chromosomal contacts.

  4. Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

    Science.gov (United States)

    Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

    2011-01-01

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

  5. Automatic Event Detection and Characterization of solar events with IRIS, SDO/AIA and Hi-C

    Science.gov (United States)

    Alexander, Caroline; Fayock, Brian; Winebarger, Amy

    2016-05-01

    Dynamic, low-lying loops with peak temperatures data due to some lower temperature spectral lines in the passbands, but have not been studied in great detail. We have developed a technique to automatically identify events (i.e., brightenings) on a pixel-by-pixel basis applying a set of selection criteria. The pixels are then grouped according to their proximity in space and relative progression of the event. This method allows us to characterize their overall lifetime and the rate at which these events occur. Our current progress includes identification of these groups of events in IRIS data, determination of their existence in AIA data, and characterization based on a comparison between the two. This technique has also been used on Hi-C data in preparation for the rocket re-flight in July 2016. Results on the success of this technique at identifying real structures and sources of heating will be shown.

  6. HiFive: a tool suite for easy and efficient HiC and 5C data analysis.

    Science.gov (United States)

    Sauria, Michael Eg; Phillips-Cremins, Jennifer E; Corces, Victor G; Taylor, James

    2015-10-24

    The chromatin interaction assays 5C and HiC have advanced our understanding of genomic spatial organization, but analysis approaches for these data are limited by usability and flexibility. The HiFive tool suite provides efficient data handling and a variety of normalization approaches for easy, fast analysis and method comparison. Integration of MPI-based parallelization allows scalability and rapid processing time. In addition to single-command analysis of an entire experiment from mapped reads to interaction values, HiFive has been integrated into the open-source, web-based platform Galaxy to connect users with computational resources and a graphical interface. HiFive is open-source software available from http://taylorlab.org/software/hifive/ .

  7. AtREM1, a Member of a New Family of B3 Domain-Containing Genes, Is Preferentially Expressed in Reproductive Meristems1

    Science.gov (United States)

    Franco-Zorrilla, José M.; Cubas, Pilar; Jarillo, José A.; Fernández-Calvín, Begoña; Salinas, Julio; Martínez-Zapater, José M.

    2002-01-01

    We have isolated and characterized AtREM1, the Arabidopsis ortholog of the cauliflower (Brassica oleracea) BoREM1. AtREM1 belongs to a large gene family of more than 20 members in Arabidopsis. The deduced AtREM1 protein contains several repeats of a B3-related domain, and it could represent a new class of regulatory proteins only found in plants. Expression of AtREM1 is developmentally regulated, being first localized in a few central cells of vegetative apical meristems, and later expanding to the whole inflorescence meristem, as well as primordia and organs of third and fourth floral whorls. This specific expression pattern suggests a role in the organization of reproductive meristems, as well as during flower organ development. PMID:11842146

  8. Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultaet fuer Physik, Universitaet Regensburg, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Stierl, M.; Hegemann, P. [Institut fuer Biologie/Experimentelle Biophysik, Humboldt Universitaet zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany); Kateriya, Suneel [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India)

    2011-08-25

    Graphical abstract: The photoactivated adenylyl cyclase (nPAC) from Naegleria gruberi was expressed heterologously in Escherichia coli and its photo-cycling dynamics was studied by optical absorption and fluorescence spectroscopy. Highlights: {yields} Photo-activated adenylyl cyclase (nPAC) from Naegleria gruberi NEG-M was expressed. {yields} Photodynamics of BLUF domain in BLUF sensor - cyclase actuator protein was studied. {yields} Photo-excitation caused BLUF photo-cycling and permanent protein re-conformation. {yields} Re-conformed protein enabled photo-induced flavin reduction by proton transfer. {yields} Fluorescence of flavin in dark- and light-adapted state of nPAC was characterized. - Abstract: The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF (blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl{sub ox}) to semi-reduced flavin (FlH{sup {center_dot}}) occurred. Furthermore, photo-excitation of FlH{sup {center_dot}} caused irreversible electron transfer to fully reduced anionic flavin (FlH{sup -}). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr{sup {center_dot}+}-Fl{sup {center_dot}-} or Trp{sup {center_dot}+}-Fl{sup {center_dot}-} radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl{sub ox} to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.

  9. Identification, molecular cloning and expression analysis of a HORMA domain containing Autophagy-related gene 13 (ATG13 from the coleopteran beetle, Tenebrio molitor

    Directory of Open Access Journals (Sweden)

    Jung Hee eLee

    2015-06-01

    Full Text Available Autophagy is a process that is necessary during starvation as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg proteins that manifest in the nucleation, elongation, and curving of autophagosome membrane. We isolated a homolog of an ATG13 gene from the transcriptome database of the larva of the mealworm beetle, Tenebrio molitor (designated as TmATG13. The sequence analysis showed that TmATG13 cDNA comprises of 1,176 bp open reading frame that encodes a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region, rich in regulatory phosphorylation sites. The N-terminal Atg13 domain show a HORMA (Hop1, Rev7, and Mad2 fold containing conserved amino acid residues across the Atg13 orthologs in insects. qRT-PCR revealed that TmATG13 was expressed ubiquitously in all the developmental stages of insect. TmATG13 mRNA expression was high in fat body and gut of the larval and adult stages of the insect. During ovary development and maturation, the TmATG13 transcripts showed high expression until six days of development, followed by a significant decline. The prospective functions mediated by TmAtg13 during autophagy will be clarified by further studies in the near future.

  10. CHiCP: a web-based tool for the integrative and interactive visualization of promoter capture Hi-C datasets.

    Science.gov (United States)

    Schofield, E C; Carver, T; Achuthan, P; Freire-Pritchett, P; Spivakov, M; Todd, J A; Burren, O S

    2016-08-15

    Promoter capture Hi-C (PCHi-C) allows the genome-wide interrogation of physical interactions between distal DNA regulatory elements and gene promoters in multiple tissue contexts. Visual integration of the resultant chromosome interaction maps with other sources of genomic annotations can provide insight into underlying regulatory mechanisms. We have developed Capture HiC Plotter (CHiCP), a web-based tool that allows interactive exploration of PCHi-C interaction maps and integration with both public and user-defined genomic datasets. CHiCP is freely accessible from www.chicp.org and supports most major HTML5 compliant web browsers. Full source code and installation instructions are available from http://github.com/D-I-L/django-chicp ob219@cam.ac.uk. © The Author 2016. Published by Oxford University Press. All rights reserved.

  11. Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

    Science.gov (United States)

    Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

    2018-01-01

    Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  12. 4Cin: A computational pipeline for 3D genome modeling and virtual Hi-C analyses from 4C data.

    Science.gov (United States)

    Irastorza-Azcarate, Ibai; Acemel, Rafael D; Tena, Juan J; Maeso, Ignacio; Gómez-Skarmeta, José Luis; Devos, Damien P

    2018-03-01

    The use of 3C-based methods has revealed the importance of the 3D organization of the chromatin for key aspects of genome biology. However, the different caveats of the variants of 3C techniques have limited their scope and the range of scientific fields that could benefit from these approaches. To address these limitations, we present 4Cin, a method to generate 3D models and derive virtual Hi-C (vHi-C) heat maps of genomic loci based on 4C-seq or any kind of 4C-seq-like data, such as those derived from NG Capture-C. 3D genome organization is determined by integrative consideration of the spatial distances derived from as few as four 4C-seq experiments. The 3D models obtained from 4C-seq data, together with their associated vHi-C maps, allow the inference of all chromosomal contacts within a given genomic region, facilitating the identification of Topological Associating Domains (TAD) boundaries. Thus, 4Cin offers a much cheaper, accessible and versatile alternative to other available techniques while providing a comprehensive 3D topological profiling. By studying TAD modifications in genomic structural variants associated to disease phenotypes and performing cross-species evolutionary comparisons of 3D chromatin structures in a quantitative manner, we demonstrate the broad potential and novel range of applications of our method.

  13. Tandem repeat variation near the HIC1 (hypermethylated in cancer 1) promoter predicts outcome of oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer.

    Science.gov (United States)

    Okazaki, Satoshi; Schirripa, Marta; Loupakis, Fotios; Cao, Shu; Zhang, Wu; Yang, Dongyun; Ning, Yan; Berger, Martin D; Miyamoto, Yuji; Suenaga, Mitsukuni; Iqubal, Syma; Barzi, Afsaneh; Cremolini, Chiara; Falcone, Alfredo; Battaglin, Francesca; Salvatore, Lisa; Borelli, Beatrice; Helentjaris, Timothy G; Lenz, Heinz-Josef

    2017-11-15

    The hypermethylated in cancer 1/sirtuin 1 (HIC1/SIRT1) axis plays an important role in regulating the nucleotide excision repair pathway, which is the main oxaliplatin-induced damage-repair system. On the basis of prior evidence that the variable number of tandem repeat (VNTR) sequence located near the promoter lesion of HIC1 is associated with HIC1 gene expression, the authors tested the hypothesis that this VNTR is associated with clinical outcome in patients with metastatic colorectal cancer who receive oxaliplatin-based chemotherapy. Four independent cohorts were tested. Patients who received oxaliplatin-based chemotherapy served as the training cohort (n = 218), and those who received treatment without oxaliplatin served as the control cohort (n = 215). Two cohorts of patients who received oxaliplatin-based chemotherapy were used for validation studies (n = 176 and n = 73). The VNTR sequence near HIC1 was analyzed by polymerase chain reaction analysis and gel electrophoresis and was tested for associations with the response rate, progression-free survival, and overall survival. In the training cohort, patients who harbored at least 5 tandem repeats (TRs) in both alleles had a significantly shorter PFS compared with those who had fewer than 4 TRs in at least 1 allele (9.5 vs 11.6 months; hazard ratio, 1.93; P = .012), and these findings remained statistically significant after multivariate analysis (hazard ratio, 2.00; 95% confidence interval, 1.13-3.54; P = .018). This preliminary association was confirmed in the validation cohort, and patients who had at least 5 TRs in both alleles had a worse PFS compared with the other cohort (7.9 vs 9.8 months; hazard ratio, 1.85; P = .044). The current findings suggest that the VNTR sequence near HIC1 could be a predictive marker for oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer. Cancer 2017;123:4506-14. © 2017 American Cancer Society. © 2017 American Cancer Society.

  14. Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

    Science.gov (United States)

    2014-01-01

    Background Immune responses are generally impaired in aged mammals. T cells have been extensively studied in this context due to the initial discovery of their reduced proliferative capacity with aging. The decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery for T cell activation. Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that participate in the regulation of the early steps of T cell activation. Such conditions tip the immune balance in favor of altered T cell activation and a related decreased response in aging. Results We present evidence that the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. We used tyrosine-specific mAbs and Western blot analysis to show that a deregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Y505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels approaching those of young donors. Significant differences in the active (Y394) and inactive (Y505) phosphorylation sites of Lck in response to T cell activation were observed in elderly donors as compared to young subjects, independently of CD45 isoform expression. Conclusions Our data suggest that the role of SHP-1 in T cell

  15. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 exposed renal ischemia/reperfusion injury in rats

    Directory of Open Access Journals (Sweden)

    Zhiming Li

    2015-01-01

    Full Text Available This study was designed to evaluate whether NADPH oxidase inhibitor (apocynin preconditioning induces expression of Src homology-2 domain-containing phosphatase-1 (SHP-1 to protect against renal ischemia/reperfusion (I/R injury (RI/RI in rats. Rats were pretreated with 50 mg/kg apocynin, then subjected to 45 min ischemia and 24 h reperfusion. The results indicated that apocynin preconditioning improved the recovery of renal function and nitroso-redox balance, reduced oxidative stress injury and inflammation damage, and upregulated expression of SHP-1 as compared to RI/RI group. Therefore our study demonstrated that apocynin preconditioning provided a protection to the kidney against I/R injury in rats partially through inducing expression of SHP-1.

  16. Hic-Et-Nunc (Here-and-Now Encoding of a Moving Target for its Saccadic Foveation

    Directory of Open Access Journals (Sweden)

    Laurent Goffart

    2012-10-01

    Full Text Available The neural representation of a moving target undergoes a spatiotemporal “diffusion” while the associated retinal activity propagates toward the motor centers and recruits the appropriate muscles for its interception in the external world. Indeed, the divergent projections within the visual system and the transmissions of signals through multiple relays, with diverse conduction velocities and integration times, lead to activities that are spatially and temporally distributed across several brain regions. In spite of this neural “blurring”, accurate saccadic eye movements can be made to bring the image of a moving target onto the fovea. Such a performance indicates that the brain is able to rapidly estimate the current spatiotemporal coordinates of the target, at least at the time of saccade landing. We tested in the monkey the robustness of this estimate when a change in eye position and a delay are experimentally added before the animal launches a saccade toward a moving target and in the absence of visual feedback. These spatiotemporal perturbations were induced by a brief microstimulation in the deep superior colliculus. The results show that the interceptive saccades can remain accurate and relatively independent of the time taken to react and to foveate the target. We propose that the brain builds an estimate of the expected and current spatiotemporal (hic-et-nunc coordinates of the target and that this signal feeds the same local feedback loop as the mechanism proposed for guiding saccades toward a stationary target (Fleuriet and Goffart, 2012 Journal of Neuroscience 32 452–461.

  17. FastHiC: a fast and accurate algorithm to detect long-range chromosomal interactions from Hi-C data.

    Science.gov (United States)

    Xu, Zheng; Zhang, Guosheng; Wu, Cong; Li, Yun; Hu, Ming

    2016-09-01

    How chromatin folds in three-dimensional (3D) space is closely related to transcription regulation. As powerful tools to study such 3D chromatin conformation, the recently developed Hi-C technologies enable a genome-wide measurement of pair-wise chromatin interaction. However, methods for the detection of biologically meaningful chromatin interactions, i.e. peak calling, from Hi-C data, are still under development. In our previous work, we have developed a novel hidden Markov random field (HMRF) based Bayesian method, which through explicitly modeling the non-negligible spatial dependency among adjacent pairs of loci manifesting in high resolution Hi-C data, achieves substantially improved robustness and enhanced statistical power in peak calling. Superior to peak callers that ignore spatial dependency both methodologically and in performance, our previous Bayesian framework suffers from heavy computational costs due to intensive computation incurred by modeling the correlated peak status of neighboring loci pairs and the inference of hidden dependency structure. In this work, we have developed FastHiC, a novel approach based on simulated field approximation, which approximates the joint distribution of the hidden peak status by a set of independent random variables, leading to more tractable computation. Performance comparisons in real data analysis showed that FastHiC not only speeds up our original Bayesian method by more than five times, bus also achieves higher peak calling accuracy. FastHiC is freely accessible at:http://www.unc.edu/∼yunmli/FastHiC/ CONTACTS: : yunli@med.unc.edu or ming.hu@nyumc.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. High Throughput Identification, Purification and Structural Characterization of Water Soluble Protein Complexes in Desulfovibrio vulgaris

    Energy Technology Data Exchange (ETDEWEB)

    Dong,, Ming; Han, Bong-Gyoon; Liu, Hui-Hai; Malik, J.; Geller, Jil; Yang, Li; Choi, M.; Chandonia, John-Marc; Arbelaez, Pablo; Sterling, H. J.; Typke, Dieter; Shatsky, Max; Brenner, Steve; Fisher, Susan; Williams, Evan; Szakal, Evelin; Allen, S.; Hall, S. C.; Hazen, Terry; Witkowska, H. E.; Jin, Jiming; Glaeser, Robert; Biggin, Mark

    2010-05-17

    Our scheme for the tagless purification of water soluble complexes. 10 g of protein from a crude bacterial extract is first fractionated by ammonium sulfate precipitation and then by a series of chromatographic steps: anion exchange (IEX), hydrophobic interaction (HIC), and finally size exclusion (Gel Filtration). Fractions from the last chromatography step are trypsin digested and peptides labeled with iTRAQ reagents to allow multiplexing and quantitation during mass spectrometric analysis. Elution profiles of identified proteins are then subjected to clustering analysis.

  19. The scaling features of the 3D organization of chromosomes are highlighted by a transformation à la Kadanoff of Hi-C data

    Science.gov (United States)

    Chiariello, Andrea M.; Bianco, Simona; Annunziatella, Carlo; Esposito, Andrea; Nicodemi, Mario

    2017-11-01

    Technologies such as Hi-C and GAM have revealed that chromosomes are not randomly folded into the nucleus of cells, but are composed by a sequence of contact domains (TADs), each typically 0.5 Mb long. However, the larger scale organization of the genome remains still not well understood. To investigate the scaling behaviour of chromosome folding, here we apply an approach à la Kadanoff, inspired by the Renormalization Group theory, to Hi-C interaction data, across different cell types and chromosomes. We find that the genome is characterized by complex scaling features, where the average size of contact domains exhibits a power-law behaviour with the rescaling level. That is compatible with the existence of contact domains extending across length scales up to chromosomal sizes. The scaling exponent is statistically indistinguishable among the different murine cell types analysed. These results point toward a scenario of a universal higher-order spatial architecture of the genome, which could reflect fundamental, organizational principles.

  20. The major-effect quantitative trait locus CsARN6.1 encodes an AAA ATPase domain-containing protein that is associated with waterlogging stress tolerance by promoting adventitious root formation

    Science.gov (United States)

    In plants, the formation of hypocotyl-derived adventitious roots (AR) is an important morphological acclimation to waterlogging stress, but its genetic basis is largely unknown. In the present study, with combined use of bulked segregant analysis-based high throughput next-gen whole genome sequencin...

  1. Discrepancy between low levels of mTOR activity and high levels of p-S6 in primary central nervous system lymphoma may be explained by PAS domain-containing serine/threonine-protein kinase-mediated phosphorylation

    DEFF Research Database (Denmark)

    Marosvari, Dora; Nagy, Noemi; Kriston, Csilla

    2018-01-01

    The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6...... phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4EBP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1......, and PASK antibodies. Finally, we examined the impact of PASK inhibition on S6 phosphorylation on BHD1 cell line. mTOR-pathway activity was significantly less frequent in PCNSL compared with DLBCL. p-S6 positivity was related to mTOR-pathway in DLBCL, but not in PCNSL. Among the other kinases potentially...

  2. Infrastructure for genomic interactions: Bioconductor classes for Hi-C, ChIA-PET and related experiments [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Aaron T. L. Lun

    2016-06-01

    Full Text Available The study of genomic interactions has been greatly facilitated by techniques such as chromatin conformation capture with high-throughput sequencing (Hi-C. These genome-wide experiments generate large amounts of data that require careful analysis to obtain useful biological conclusions. However, development of the appropriate software tools is hindered by the lack of basic infrastructure to represent and manipulate genomic interaction data. Here, we present the InteractionSet package that provides classes to represent genomic interactions and store their associated experimental data, along with the methods required for low-level manipulation and processing of those classes. The InteractionSet package exploits existing infrastructure in the open-source Bioconductor project, while in turn being used by Bioconductor packages designed for higher-level analyses. For new packages, use of the functionality in InteractionSet will simplify development, allow access to more features and improve interoperability between packages.

  3. Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

    DEFF Research Database (Denmark)

    Whiteford, James R; Xian, Xiaojie; Chaussade, Claire

    2011-01-01

    Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is ß1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine p...

  4. Trichloroacetimidates as Alkylating Reagents and Their Application in the Synthesis of Pyrroloindoline Natural Products and Synthesis of Small Molecule Inhibitors of Src Homology 2 Domain-Containing Inositol Phosphatase (SHIP)

    Science.gov (United States)

    Adhikari, Arijit A.

    was applied towards the synthesis of natural products and their analogs. The pyrroloindoline ring system is found in many alkaloids and cyclic peptides which mainly differ in the substitution at the C3a position. To provide rapid access to these natural products a diversity-oriented strategy was established via displacement of C3a-trichloroacetimidate pyrroloindoline. Carbon, oxygen, sulfur and nitrogen nucleophiles were all shown to undergo substitution reactions with these trichloroacetimidates in the presence of a Lewis acid catalyst. In order to demonstrate the utility of this new method it was applied towards the synthesis of arundinine and a formal synthesis of psychotriasine. Current investigations involve the application of this method towards the synthesis of a complex pyrroloindoline natural product kapakahine C and the progress made therein has been discussed. The reactivity of trichloroacetimidates was also investigated for the selective C3-alkylation of 2,3-disubstituted indoles to provide indolenines. Indolenines serve as useful intermediates in the synthesis of many complex alkaloids. Different benzylic and allylic trichloroacetimidates were shown to provide 3,3'-disubstituted indolenines with high yields in the presence of catalytic amounts of Lewis acids. Various substituted indoles were evaluated under these reaction conditions. This methodology was also applied towards the synthesis of the core tetracyclic ring system found in communesin natural products. In addition to the above work, synthesis of small molecule inhibitors of Src Homology 2 Domain-Containing Inositol Phosphatase (SHIP) has also been described. Aberrations in the phosphoinositide 3-kinase (PI3K) cellular signaling pathway can lead to diseased cellular states like cancer. Herein we have reported stereoselective synthesis of two quinoline based small molecule SHIP inhibitors. The lead compounds and their analogs were tested for their activities against SHIP by Malachite green assay

  5. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    and phosphatases, specific sets of ubiquitinating/deubiquitinating enzymes control the degree of ubiquitination. A large number of ubiquitin-binding proteins act at different steps in the downstream pathways, followed by the ubiquitinated protein. Different families of ubiquitin-binding proteins have been...... described. UBA (ubiquitin-associated) domain-containing proteins is the largest family and includes members involved in different cell processes. The smaller groups of UIM (ubiquitin-interacting motif), GAT [GGA (Golgi-associated gamma-adaptin homologous) and Tom1 (target of Myb 1)], CUE (coupling...

  6. Introduction to the project VAHINE: VAriability of vertical and tropHIc transfer of diazotroph derived N in the south wEst Pacific

    Science.gov (United States)

    Bonnet, Sophie; Moutin, Thierry; Rodier, Martine; Grisoni, Jean-Michel; Louis, Francis; Folcher, Eric; Bourgeois, Bertrand; Boré, Jean-Michel; Renaud, Armelle

    2016-05-01

    On the global scale, N2 fixation provides the major external source of reactive nitrogen to the surface ocean, surpassing atmospheric and riverine inputs, and sustains ˜ 50 % of new primary production in oligotrophic environments. The main goal of the VAriability of vertical and tropHIc transfer of diazotroph derived N in the south wEst Pacific (VAHINE) project was to study the fate of nitrogen newly fixed by diazotrophs (or diazotroph-derived nitrogen) in oceanic food webs, and how it impacts heterotrophic bacteria, phytoplankton and zooplankton dynamics, stocks and fluxes of biogenic elements and particle export. Three large-volume ( ˜ 50 m3) mesocosms were deployed in a tropical oligotrophic ecosystem (the New Caledonia lagoon, south-eastern Pacific) and intentionally fertilized with ˜ 0.8 µM of dissolved inorganic phosphorus (DIP) to stimulate diazotrophy and follow subsequent ecosystem changes. VAHINE was a multidisciplinary project involving close collaborations between biogeochemists, molecular ecologist, chemists, marine opticians and modellers. This introductory paper describes in detail the scientific objectives of the project as well as the implementation plan: the mesocosm description and deployment, the selection of the study site (New Caledonian lagoon), and the logistical and sampling strategy. The main hydrological and biogeochemical conditions of the study site before the mesocosm deployment and during the experiment itself are described, and a general overview of the papers published in this special issue is presented.

  7. Protein: FBA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA6 vesicular transport COL4A3BP CERT, STARD11 COL4A3BP Collagen type IV alpha-3-binding pro...tein Ceramide transfer protein, Goodpasture antigen-binding protein, START domain-containing prote...in 11, StAR-related lipid transfer protein 11 9606 Homo sapiens Q9Y5P4 10087 2E3N, 2Z9Y, 2E3Q, 2Z9Z, 2E3O, 2E3R, 2E3S, 2E3P 2E3M 3H3R, 3H3Q, 3H3S, 3H3T 10087 Q9Y5P4 20036255 ...

  8. Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins

    NARCIS (Netherlands)

    Welsh, M.; Songyang, Z.; Frantz, J. D.; Trüb, T.; Reedquist, K. A.; Karlsson, T.; Miyazaki, M.; Cantley, L. C.; Band, H.; Shoelson, S. E.

    1998-01-01

    Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of

  9. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Directory of Open Access Journals (Sweden)

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  10. Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.)

    Science.gov (United States)

    Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

    2017-01-01

    Coconut (Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae (Palmaceae). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1. Its transcriptional activities and interactions with the acetyl-CoA carboxylase (BCCP2) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis, high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase (P < 0.05) in seed oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined. PMID:28179911

  11. Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.).

    Science.gov (United States)

    Sun, RuHao; Ye, Rongjian; Gao, Lingchao; Zhang, Lin; Wang, Rui; Mao, Ting; Zheng, Yusheng; Li, Dongdong; Lin, Yongjun

    2017-01-01

    Coconut ( Cocos nucifera L.) is a key tropical crop and a member of the monocotyledonous family Arecaceae ( Palmaceae ). Few genes and related metabolic processes involved in coconut endosperm development have been investigated. In this study, a new member of the WRI1 gene family was isolated from coconut endosperm and was named CoWRI1 . Its transcriptional activities and interactions with the acetyl-CoA carboxylase ( BCCP2 ) promoter of CoWRI1 were confirmed by the yeast two-hybrid and yeast one-hybrid approaches, respectively. Functional characterization was carried out through seed-specific expression in Arabidopsis and endosperm-specific expression in rice. In transgenic Arabidopsis , high over-expressions of CoWRI1 in seven independent T2 lines were detected by quantitative real-time PCR. The relative mRNA accumulation of genes encoding enzymes involved in either fatty acid biosynthesis or triacylglycerols assembly (BCCP2, KASI, MAT, ENR, FATA, and GPDH) were also assayed in mature seeds. Furthermore, lipid and fatty acids C16:0 and C18:0 significantly increased. In two homozygous T2 transgenic rice lines (G5 and G2), different CoWRI1 expression levels were detected, but no CoWRI1 transcripts were detected in the wild type. Analyses of the seed oil content, starch content, and total protein content indicated that the two T2 transgenic lines showed a significant increase ( P oil content. The transgenic lines also showed a significant increase in starch content, whereas total protein content decreased significantly. Further analysis of the fatty acid composition revealed that palmitic acid (C16:0) and linolenic acid (C18:3) increased significantly in the seeds of the transgenic rice lines, but oleic acid (C18:1) levels significantly declined.

  12. A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

    Science.gov (United States)

    Yu, Shoukai; Lemos, Bernardo

    2016-12-31

    Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  13. Prediction of protein retention times in hydrophobic interaction chromatography by robust statistical characterization of their atomic-level surface properties.

    Science.gov (United States)

    Hanke, Alexander T; Klijn, Marieke E; Verhaert, Peter D E M; van der Wielen, Luuk A M; Ottens, Marcel; Eppink, Michel H M; van de Sandt, Emile J A X

    2016-03-01

    The correlation between the dimensionless retention times (DRT) of proteins in hydrophobic interaction chromatography (HIC) and their surface properties were investigated. A ternary atomic-level hydrophobicity scale was used to calculate the distribution of local average hydrophobicity across the proteins surfaces. These distributions were characterized by robust descriptive statistics to reduce their sensitivity to small changes in the three-dimensional structure. The applicability of these statistics for the prediction of protein retention behaviour was looked into. A linear combination of robust statistics describing the central tendency, heterogeneity and frequency of highly hydrophobic clusters was found to have a good predictive capability (R2  = 0.78), when combined a factor to account for protein size differences. The achieved error of prediction was 35% lower than for a similar model based on a description of the protein surface on an amino acid level. This indicates that a robust and mathematically simple model based on an atomic description of the protein surface can be used for the prediction of the retention behaviour of conformationally stable globular proteins with a well determined 3D structure in HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:372-381, 2016. © 2016 American Institute of Chemical Engineers.

  14. Microtubule-Associated Protein Mdp3 Promotes Breast Cancer Growth and Metastasis

    OpenAIRE

    Tala,; Xie, Songbo; Sun, Xiaodong; Sun, Xiaoou; Ran, Jie; Zhang, Linlin; Li, Dengwen; Liu, Min; Bao, Gang; Zhou, Jun

    2014-01-01

    Breast cancer is the most prevalent cancer in women worldwide with a high mortality rate, and the identification of new biomarkers and targets for this disease is greatly needed. Here we present evidence that microtubule-associated protein (MAP) 7 domain-containing protein 3 (Mdp3) is highly expressed in clinical samples and cell lines of breast cancer. The expression of Mdp3 correlates with clinicopathological parameters indicating breast cancer malignancy. In addition, Mdp3 promotes breast ...

  15. Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins

    OpenAIRE

    Meng, Xiangzhi; Krumm, Brian; Li, Yongchao; Deng, Junpeng; Xiang, Yan

    2015-01-01

    Productive viral replication requires overcoming many barriers posed by the host innate immune system. Human sterile alpha motif domain-containing 9 (SAMD9) is a newly identified antiviral factor that is specifically targeted by poxvirus proteins belonging to the C7 family of host-range factors. Here we provide the first, to our knowledge, atomic view of two functionally divergent proteins from the C7 family and determine the molecular basis that dictates whether they can target SAMD9 effecti...

  16. Small angle X-ray scattering and transmission electron microscopy study of the Lactobacillus brevis S-layer protein

    Energy Technology Data Exchange (ETDEWEB)

    Jaeaeskelaeinen, Pentti [Department of Biomedical Engineering and Computational Science, PO Box 2200, FI-02015 Aalto University School of Science and Technology (Finland); Engelhardt, Peter [Haartman Institute, Department of Pathology, PO Box 21, FIN-00014 University of Helsinki (Finland); Hynoenen, Ulla; Palva, Airi [Department of Basic Veterinary Sciences, Division of Microbiology, FIN-00014 University of Helsinki (Finland); Torkkeli, Mika; Serimaa, Ritva, E-mail: ritva.serimaa@helsinki.f [Department of Physics, POB 64, 00014 University of Helsinki (Finland)

    2010-10-01

    The structure of self-assembly domain containing recombinant truncation mutants of Lactobacillus brevis surface layer protein SlpA in aqueous solution was studied using small-angle X-ray scattering and transmission electron microscopy. The proteins were found out to interact with each other forming stable globular oligomers of about 10 monomers. The maximum diameter of the oligomers varied between 75 A and 435 A.

  17. Small angle X-ray scattering and transmission electron microscopy study of the Lactobacillus brevis S-layer protein

    Science.gov (United States)

    Jääskeläinen, Pentti; Engelhardt, Peter; Hynönen, Ulla; Torkkeli, Mika; Palva, Airi; Serimaa, Ritva

    2010-10-01

    The structure of self-assembly domain containing recombinant truncation mutants of Lactobacillus brevis surface layer protein SlpA in aqueous solution was studied using small-angle X-ray scattering and transmission electron microscopy. The proteins were found out to interact with each other forming stable globular oligomers of about 10 monomers. The maximum diameter of the oligomers varied between 75 Å and 435 Å.

  18. Ubiquitin-binding proteins: similar, but different

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Hofmann, Kay; Hartmann-Petersen, Rasmus

    2005-01-01

    of ubiquitin conjugation to endoplasmic reticulum degradation), UEV [ubiquitin E2 (ubiquitin-conjugating enzyme) variant] and NZF (nuclear protein localization gene 4 zinc finger) domain-containing proteins appear to have more specialized functions. Here we discuss functional and structural properties......Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Typically, ubiquitinated proteins are targeted for degradation by the 26 S proteasome. However, more recently the ubiquitin signal has also been connected with many other cell processes, including...... endocytosis, vesicle fusion, DNA repair and transcriptional silencing. Hence ubiquitination may be comparable with phosphorylation in its importance as an intracellular switch, controlling various signal-transduction pathways. Similar to the regulation of the extent of phosphorylation by kinases...

  19. WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

    Science.gov (United States)

    Chen, Shuai; Wang, Han; Huang, Yu-Fan; Li, Ming-Li; Cheng, Jiang-Hong; Hu, Peng; Lu, Chuan-Hui; Zhang, Ya; Liu, Na; Tzeng, Chi-Meng; Zhang, Zhi-Ming

    2017-07-19

    The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

  20. Poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monolithic column for efficient hydrophobic interaction chromatography of proteins.

    Science.gov (United States)

    Li, Yuanyuan; Tolley, H Dennis; Lee, Milton L

    2009-11-15

    Rigid poly[hydroxyethyl acrylate-co-poly(ethylene glycol) diacrylate] monoliths were synthesized inside 75 mum i.d. capillaries by one-step UV-initiated copolymerization using methanol and ethyl ether as porogens. The optimized monolithic column was evaluated for hydrophobic interaction chromatography (HIC) of standard proteins. Six proteins were separated within 20 min with high resolution using a 20 min elution gradient, resulting in a peak capacity of 54. The effect of gradient rate and initial salt concentration on the retention of proteins were investigated. Mass recovery was found to be greater than 96%, indicating the biocompatibility of this monolith. The monolith was mechanically stable and showed nearly no swelling or shrinking in different polarity solvents. The preparation of this in situ polymerized acrylate monolithic column was highly reproducible. The run-to-run and column-to-column reproducibilities were less than 2.0% relative standard deviation (RSD) on the basis of the retention times of protein standards. The performance of this monolithic column for HIC was comparable or superior to the performance of columns packed with small particles.

  1. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  2. The SNARE protein family of Leishmania major

    Directory of Open Access Journals (Sweden)

    Mottram Jeremy C

    2006-10-01

    Full Text Available Abstract Background Leishmania major is a protozoan parasite with a highly polarised cell shape that depends upon endocytosis and exocytosis from a single area of the plasma membrane, the flagellar pocket. SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors are key components of the intracellular vesicle-mediated transports that take place in all eukaryotic cells. They are membrane-bound proteins that facilitate the docking and fusion of vesicles with organelles. The recent availability of the genome sequence of L. major has allowed us to assess the complement of SNAREs in the parasite and to investigate their location in comparison with metazoans. Results Bioinformatic searches of the L. major genome revealed a total of 27 SNARE domain-containing proteins that could be classified in structural groups by phylogenetic analysis. 25 of these possessed the expected features of functional SNAREs, whereas the other two could represent kinetoplastid-specific proteins that might act as regulators of the SNARE complexes. Other differences of Leishmania SNAREs were the absence of double SNARE domain-containing and of the brevin classes of these proteins. Members of the Qa group of Leishmania SNAREs showed differential expressions profiles in the two main parasite forms whereas their GFP-tagging and in vivo expression revealed localisations in the Golgi, late endosome/lysosome and near the flagellar pocket. Conclusion The early-branching eukaryote L. major apparently possess a SNARE repertoire that equals in number the one of metazoans such as Drosophila, showing that the machinery for vesicle fusion is well conserved throughout the eukaryotes. However, the analysis revealed the absence of certain types of SNAREs found in metazoans and yeast, while suggesting the presence of original SNAREs as well as others with unusual localisation. This study also presented the intracellular localisation of the L. major SNAREs from the Qa group

  3. FYVE-dependent endosomal targeting of an arrestin-related protein in amoeba.

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    Dorian Guetta

    Full Text Available BACKGROUND: Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.

  4. Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

    Science.gov (United States)

    Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

    2014-09-01

    Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. IrML – a gene encoding a new member of the ML protein family from the hard tick, Ixodes ricinus

    Czech Academy of Sciences Publication Activity Database

    Horáčková, J.; Rudenko, Natalia; Golovchenko, Maryna; Havlíková, S.; Grubhoffer, Libor

    2010-01-01

    Roč. 35, č. 2 (2010), s. 410-418 ISSN 1081-1710 R&D Projects: GA ČR(CZ) GA524/06/1479; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518 Keywords : Ixodes ricinus * tick * ML-domain containing protein * in situ hybridization * gene expression * ML (MD-2-related lipid-recognition) domain Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 1.256, year: 2010

  6. TAD-free analysis of architectural proteins and insulators.

    Science.gov (United States)

    Mourad, Raphaël; Cuvier, Olivier

    2017-12-19

    The three-dimensional (3D) organization of the genome is intimately related to numerous key biological functions including gene expression and DNA replication regulations. The mechanisms by which molecular drivers functionally organize the 3D genome, such as topologically associating domains (TADs), remain to be explored. Current approaches consist in assessing the enrichments or influences of proteins at TAD borders. Here, we propose a TAD-free model to directly estimate the blocking effects of architectural proteins, insulators and DNA motifs on long-range contacts, making the model intuitive and biologically meaningful. In addition, the model allows analyzing the whole Hi-C information content (2D information) instead of only focusing on TAD borders (1D information). The model outperforms multiple logistic regression at TAD borders in terms of parameter estimation accuracy and is validated by enhancer-blocking assays. In Drosophila, the results support the insulating role of simple sequence repeats and suggest that the blocking effects depend on the number of repeats. Motif analysis uncovered the roles of the transcriptional factors pannier and tramtrack in blocking long-range contacts. In human, the results suggest that the blocking effects of the well-known architectural proteins CTCF, cohesin and ZNF143 depend on the distance between loci, where each protein may participate at different scales of the 3D chromatin organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Estudio de la susceptibilidad de un acero inoxidable dúplex del tipo 22Cr5NiMoN al dañado por hidrógeno en condiciones estáticas (HIC y bajo carga (SSC

    Directory of Open Access Journals (Sweden)

    Gutiérrez de Saiz-Solabarría, S.

    1998-05-01

    Full Text Available The behavior to hydrogen damage caused by corrosion in a H2S medium is studied in a molded ferrite- austenite (52-48 % duplex stainless steel 22Cr5NiMoN type (UNS-J9.22.05 under both, static (damaging mechanism called Hydrogen Induced Cracking (HIC and sustained load (damaging mechanism called Sulfide Stress Cracking (SSC, conditions.

    Se estudia el comportamiento de un mismo acero moldeado inoxidable dúplex austeno-ferrítico (48-52 % del tipo 22Cr5NiMoN (UNS-J9.22.05 frente al dañado por hidrógeno generado por corrosión en medio H2S, tanto en condiciones estáticas, mecanismo de dañado conocido como HIC (Hydrogen Induced Cracking, como bajo carga de tracción, mecanismo de dañado conocido como SSC (Sulfide Stress Cracking.

  9. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Directory of Open Access Journals (Sweden)

    Muhammad Ibrahim

    Full Text Available Outer membrane (OM proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  10. Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

    Science.gov (United States)

    Ibrahim, Muhammad; Shi, Yu; Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Blondel, Carlos; Santiviago, Carlos A; Contreras, Ines; Sun, Guochang

    2012-01-01

    Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

  11. Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense.

    Science.gov (United States)

    Widjaja, Ivy; Naumann, Kai; Roth, Udo; Wolf, Noreen; Mackey, David; Dangl, Jeffery L; Scheel, Dierk; Lee, Justin

    2009-01-01

    Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone-responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2-DE facilitated the discovery of low abundance proteins - enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA-binding protein, and a C2-domain-containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA-binding protein, involvement of PTM and post-transcriptional regulation are implicated, respectively.

  12. Immunohistochemical detection of the apoptosis-related proteins FADD, FLICE, and FLIP in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Bank, Micha I; Gudbrand, Charlotte; Lundegaard, Pia Rengtved

    2005-01-01

    FLICE, and FLIP. The clinical outcome of the disease could not be correlated to the expression of the investigated proteins. This study shows a high expression of the apoptosis-related proteins FADD, active FLICE, and FLIP in pLCs. The authors previously showed that pLCs express Fas and Fas ligand......Langerhans cell histiocytosis (LCH) is characterized by an accumulation of dendritic Langerhans cells in granulomatous lesions in various organs. The etiology of LCH remains enigmatic. Fas/APO-1/CD95 belongs to the "death receptor" family of apoptosis regulators and has been implicated...... in the downregulation of immune responses. The authors examined the expression of three proteins that are engaged in the Fas signaling cascade-FADD/Fas-associated death domain-containing protein, FLICE/FADD-like interleukin-1beta-converting enzyme (both pro-apoptotic), and FLIP/FLICE-inhibitory protein (anti...

  13. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  14. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  15. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    Science.gov (United States)

    Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

    2013-12-13

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

  16. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Muhammed Jamsheer K

    Full Text Available Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  17. Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

    DEFF Research Database (Denmark)

    Wilkinson, C R; Seeger, M; Hartmann-Petersen, R

    2001-01-01

    The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA...... domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S...

  18. 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

  19. Resistance to inhibitors of cholinesterase 8A catalyzes release of Gαi-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Gαi-GDP complexes

    Science.gov (United States)

    Tall, Gregory G.; Gilman, Alfred G.

    2005-01-01

    Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein α-subunits. Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have placed RIC-8 in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the Gαi class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of Gαi functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and Gαi regulate a similar process. We find that mammalian Ric-8A dissociates Gαi-GDP/LGN/NuMA complexes catalytically, releasing activated Gαi-GTP in vitro. Ric-8A-stimulated activation of Gαi caused concomitant liberation of NuMA from LGN. We conclude that Ric-8A efficiently utilizes GoLoco/Gαi-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Gαi-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division. PMID:16275912

  20. Resistance to inhibitors of cholinesterase 8A catalyzes release of Galphai-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Galphai-GDP complexes.

    Science.gov (United States)

    Tall, Gregory G; Gilman, Alfred G

    2005-11-15

    Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have placed RIC-8 in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the Galphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of Galphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and Galphai regulate a similar process. We find that mammalian Ric-8A dissociates Galphai-GDP/LGN/NuMA complexes catalytically, releasing activated Galphai-GTP in vitro. Ric-8A-stimulated activation of Galphai caused concomitant liberation of NuMA from LGN. We conclude that Ric-8A efficiently utilizes GoLoco/Galphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division.

  1. Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley

    DEFF Research Database (Denmark)

    Rayapuram, Channabasavangowda; Jensen, Michael Krogh; Maiser, Fabian

    2012-01-01

    , followed by a rather short 17-amino-acid transmembrane domain, which includes an AAA motif, two features characteristic of endoplasmic reticulum (ER)-targeted proteins and, finally, a characteristic putative protein kinase domain in the C-terminus. The HvCRK1 transcript was isolated from leaves inoculated......The receptor-like protein kinases (RLKs) constitute a large and diverse group of proteins controlling numerous plant physiological processes, including development, hormone perception and stress responses. The cysteine-rich RLKs (CRKs) represent a prominent subfamily of transmembrane-anchored RLKs....... We have identified a putative barley (Hordeum vulgare) CRK gene family member, designated HvCRK1. The mature putative protein comprises 645 amino acids, and includes a putative receptor domain containing two characteristic ‘domain 26 of unknown function’ (duf26) domains in the N-terminal region...

  2. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers......, are reported. The aim is to depict how the elucidation of the interplay of structures requires the interplay of methods....

  3. Functional conservation study of polarity protein Crumbs intracellular domain.

    Science.gov (United States)

    Shi, Qi-ping; Cao, Hao-wei; Xu, Rui; Zhang, Dan-dan; Huang, Juan

    2017-01-20

    The transmembrane protein Crumbs (Crb) plays key roles in the establishing and maintaining cell apical-basal polarity in epithelial cells by determining the apical plasma membrane identity. Although its intracellular domain contains only 37 amino acids, it is absolutely essential for its function. In Drosophila, mutations in this intracellular domain result in severe defects in epithelial polarity and abnormal embryonic development. The intracellular domain of Crb shows high homology across species from Drosophila to Mus musculus and Homo sapiens. However, the intracellular domains of the two Crb proteins in C. elegans are rather divergent from those of Drosophila and mammals, raising the question on whether the function of the intracellular domain of the Crb protein is conserved in C. elegans. Using genomic engineering approach, we replaced the intracellular domain of the Drosophila Crb with that of C. elegans Crb2 (CeCrb2), which has extremely low homology with those from the Crb proteins of Drosophila and mammals. Surprisingly, substituting the intracellular domain of Drosophila Crb with that of CeCrb2 did not cause any abnormalities in development of the Drosophila embryo, in terms of expression and localization of Crb and other polarity proteins and apical-basal polarity in embryonic epithelial cells. Our results support the notion that despite their extensive sequence variations, all functionally critical amino acid residues and motifs of the intercellular domain of Crb proteins are fully conserved between Drosophila and C. elegans.

  4. Functional genomic analysis of cassava proteins with TIR domains

    International Nuclear Information System (INIS)

    Roman Reyna, Veronica; Lopez, Camilo

    2012-01-01

    Proteins containing a TIR domain (toll interleukin receptor) are involved in plant and animal immunity. The aim of this work was to carry out an overall genomic analysis of cassava proteins with a TIR domain and discern their possible role in resistance to cassava bacterial blight. In total 46 proteins with a TIR domain were identified in the cassava proteome and were classed in four categories according the presence or absence of other domains: TIR (T), TIR -NB (TN), TIR - lRR (TL) and TIR - NB - lRR (TNL). 56.6 % of these 46 proteins have TIR, NB and lRR domains. Using multiple alignments it was possible to demonstrate that not all cassava TIR domains contain the AE region, involved in dimerization and activation of immune responses. Three of the four proteins categories (T, TNL and TN) presented a higher number of synonymous substitutions suggesting that they are not involved in recognition process. two TIR domains not presenting the ae region were analyzed by yeast two hybrid assays and by agro-infiltration, finding that both are able to form homo and heterodimers, but they do not trigger defense responses. With this study it was possible to conclude that TIR domains can function as adaptors in the signal transduction with other resistance proteins. In addition, it became clear that not always the AE region is important for TIR dimerization but it seems necessary to activate defense responses signals.

  5. Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus.

    Directory of Open Access Journals (Sweden)

    Jiwu Chen

    Full Text Available In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.

  6. A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

    Science.gov (United States)

    Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

    2014-04-16

    Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

  7. Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

    Science.gov (United States)

    Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

    2016-08-26

    Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. High-throughput oxidation screen of antibody-drug conjugates by analytical protein A chromatography following IdeS digest.

    Science.gov (United States)

    Buecheler, Jakob W; Winzer, Matthias; Weber, Christian; Gieseler, Henning

    2018-05-01

    Oxidation of protein therapeutics is a major chemical degradation pathway which may impact bioactivity, serum half-life and stability. Therefore, oxidation is a relevant parameter which has to be monitored throughout formulation development. Methods such as HIC, RPLC and LC/MS achieve a separation of oxidized and non-oxidized species by differences in hydrophobicity. Antibody-drug conjugates (ADC) although are highly more complex due to the heterogeneity in linker, drug, drug-to-antibody ratio (DAR) and conjugation site. The analytical protein A chromatography can provide a simple and fast alternative to these common methods. A miniature analytical protein A chromatography method in combination with an IdeS digest was developed to analyse ADCs. The IdeS digest efficiency of an IgG1 was monitored using SEC-HPLC and non-reducing SDS-PAGE. An antibody-fluorescent dye conjugate was conjugated at different dye-to-antibody ratios as model construct to mimic an ADC. With IdeS, an almost complete digest of a model IgG1 can be achieved (digested protein amount >98%). This enables subsequent analytical protein A chromatography, which consequently eliminates any interference of payload with the stationary phase. A novel high-throughput method for an interchain cysteine-linked ADC oxidation screens during formulation development was developed. © 2018 Royal Pharmaceutical Society.

  9. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya.

    Science.gov (United States)

    Jue, Dengwei; Sang, Xuelian; Shu, Bo; Liu, Liqin; Wang, Yicheng; Jia, Zhiwei; Zou, Yu; Shi, Shengyou

    2017-01-01

    Ripening affects the quality and nutritional contents of fleshy fruits and is a crucial process of fruit development. Although several studies have suggested that ubiquitin-conjugating enzyme (E2s or UBC enzymes) are involved in the regulation of fruit ripening, little is known about the function of E2s in papaya (Carica papaya). In the present study, we searched the papaya genome and identified 34 putative UBC genes, which were clustered into 17 phylogenetic subgroups. We also analyzed the nucleotide sequences of the papaya UBC (CpUBC) genes and found that both exon-intron junctions and sequence motifs were highly conserved among the phylogenetic subgroups. Using real-time PCR analysis, we also found that all the CpUBC genes were expressed in roots, stems, leaves, male and female flowers, and mature fruit, although the expression of some of the genes was increased or decreased in one or several specific organs. We also found that the expression of 13 and two CpUBC genes were incresesd or decreased during one and two ripening stages, respectively. Expression analyses indicates possible E2s playing a more significant role in fruit ripening for further studies. To the best of our knowledge, this is the first reported genome-wide analysis of the papaya UBC gene family, and the results will facilitate further investigation of the roles of UBC genes in fruit ripening and will aide in the functional validation of UBC genes in papaya.

  10. Structure of a WW domain-containing fragment of dystrophin complexed with {beta}-dystroglycan.

    Energy Technology Data Exchange (ETDEWEB)

    Huang, X.; Poy, F.; Zhang, R.; Joachimiak, A.; Sudol, M.; Eck, M. J.; Biosciences Division; Dana Farber Cancer Inst.; Harvard Medical School; Mount Sinai School of Medicine

    2000-08-01

    Dystrophin and {beta}-dystroglycan are components of the dystrophin--glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of {beta}-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of {beta}-dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in {beta}-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The {beta}-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.

  11. Immunohistochemical Localization of Fibrinogen C Domain Containing 1 on Epithelial and Mucosal Surfaces in Human Tissues

    DEFF Research Database (Denmark)

    von Huth, Sebastian; Moeller, Jesper B; Schlosser, Anders

    2018-01-01

    high expression of FIBCD1 and also mesodermal-derived cells in the genitourinary system and ectodermal-derived epidermis and sebaceous glands cells expressed FIBCD1. In some columnar epithelial cells, for example, in the salivary gland and gall bladder, the FIBCD1 expression was clearly polarized...

  12. The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

    Directory of Open Access Journals (Sweden)

    Enda Shevlin

    Full Text Available Toll-like receptor 7 (TLR7 plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/- murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/- cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

  13. The chicken lysozyme chromatin domain contains a second, widely expressed gene

    Science.gov (United States)

    Chong, Suyinn; Riggs, Arthur D.; Bonifer, Constanze

    2002-01-01

    The chicken lysozyme (cLys) locus has been shown to contain all of the cis-elements necessary for position-independent and tissue-specific expression entirely within a 24-kb region defined by general DNase I sensitivity and flanked by matrix attachment regions. As such, it has been viewed as an example of a functional chromatin domain, which is structurally and functionally isolated from neighbouring chromatin. We report here the identification and characterisation of the chicken glioma-amplified sequence (cGas41) locus, which though widely expressed, is contained entirely within the lysozyme chromatin domain. The cGas41 transcript encodes a putative transcription factor, starts 207 bp downstream of the cLys polyadenylation site and is preceded by a CpG island with proposed dual promoter/origin function. The location and differential expression of cGas41 compels re-evaluation of the accumulated literature on the lysozyme domain, and represents an example of two unrelated, differentially expressed vertebrate genes coexisting in the same functional chromatin domain. PMID:11788708

  14. Microtubule-associated protein Mdp3 promotes breast cancer growth and metastasis.

    Science.gov (United States)

    Tala; Xie, Songbo; Sun, Xiaodong; Sun, Xiaoou; Ran, Jie; Zhang, Linlin; Li, Dengwen; Liu, Min; Bao, Gang; Zhou, Jun

    2014-01-01

    Breast cancer is the most prevalent cancer in women worldwide with a high mortality rate, and the identification of new biomarkers and targets for this disease is greatly needed. Here we present evidence that microtubule-associated protein (MAP) 7 domain-containing protein 3 (Mdp3) is highly expressed in clinical samples and cell lines of breast cancer. The expression of Mdp3 correlates with clinicopathological parameters indicating breast cancer malignancy. In addition, Mdp3 promotes breast cancer cell proliferation and motility in vitro and stimulates breast cancer growth and metastasis in mice. Mechanistic studies reveal that γ-tubulin interacts with and recruits Mdp3 to the centrosome and that the centrosomal localization of Mdp3 is required for its activity to promote breast cancer cell proliferation and motility. These findings suggest a critical role for Mdp3 in the growth and metastasis of breast cancer and may have important implications for the management of this disease.

  15. Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST

    Directory of Open Access Journals (Sweden)

    Hongyun Nie

    2016-01-01

    Full Text Available Protocadherin 15 (PCDH15 is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23. Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

  16. SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.

    Directory of Open Access Journals (Sweden)

    Lukas von Tobel

    2014-11-01

    Full Text Available Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD syndrome.

  17. The BRCT domain is a phospho-protein binding domain.

    Science.gov (United States)

    Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

    2003-10-24

    The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

  18. Interfacial Protein-Protein Associations

    OpenAIRE

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2013-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for ...

  19. Direct protein-protein interaction between PLCγ1 and the bradykinin B2 receptor-Importance of growth conditions

    International Nuclear Information System (INIS)

    Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.

    2005-01-01

    Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells

  20. The nuclear RNA binding protein RBP33 influences mRNA and spliced leader RNA abundance in Trypanosoma brucei.

    Science.gov (United States)

    Cirovic, Olivera; Trikin, Roman; Hoffmann, Anneliese; Doiron, Nicholas; Jakob, Martin; Ochsenreiter, Torsten

    2017-03-01

    RNA recognition motif (RRM) containing proteins are important regulators of gene expression in trypanosomes. Here we expand our current knowledge on the exclusively nuclear localized RRM domain containing protein RBP33 of Trypanosoma brucei. Overexpression of RBP33 leads to a quick growth arrest in G2/M in bloodstream form cells likely due to an overall mRNA- and spliced leader abundance decrease while the ribosomal RNAs remain unaffected. The recombinant RBP33 binds to poly(A) and random sequence RNA in vitro confirming its role as a RNA binding protein. Finally super-resolution microscopy detects RBP33 in small punctae throughout the nucleus and surrounding the nucleolus, however the signal is depleted inside the nucleolus. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Caenorhabditis elegans NONO-1: Insights into DBHS protein structure, architecture, and function.

    Science.gov (United States)

    Knott, Gavin J; Lee, Mihwa; Passon, Daniel M; Fox, Archa H; Bond, Charles S

    2015-12-01

    Members of the Drosophila behavior/human splicing (DBHS) protein family have been characterized in the vertebrates Homo sapiens and Mus musculus, and the invertebrates Drosophila melanogaster and Chironomus tentans. Collectively, both vertebrate and invertebrate DBHS proteins function throughout gene regulation, largely but not always, within the nucleus. In this study, we report a structural and bioinformatic analysis of the DBHS protein family to guide future studies into DBHS protein function. To explore the structural plasticity of the family, we describe the 2.4 Å crystal structure of Caenorhabditis elegans non-POU domain-containing octamer-binding protein 1 (NONO-1). The structure is dimeric, with a domain arrangement consistent with mammalian DBHS proteins. Comparison with the DBHS structures available from H. sapiens reveals that there is inherent domain flexibility within the homologous DBHS region. Mapping amino acid similarity within the family to the NONO-1 dimer highlights the dimer interface, coiled-coil oligomerization motif, and putative RNA binding surfaces. Surprisingly, the interior surface of RNA recognition motif 2 (RRM2) that faces a large internal void is highly variable, but the external β2-β3 loops of RRM2 show remarkable preservation. Overall, the DBHS region is under strong purifying selection, whereas the sequences N- and C-terminal to the DBHS region are less constrained. The findings described in this study provide a molecular basis for further investigation into the mechanistic function of the DBHS protein family in biology. © 2015 The Protein Society.

  2. Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT protein 2.

    Directory of Open Access Journals (Sweden)

    David A Gross

    2010-05-01

    Full Text Available Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2 belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9AAA in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

  3. Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

    Science.gov (United States)

    Nezametdinova, V Z; Mavletova, D A; Alekseeva, M G; Chekalina, M S; Zakharevich, N V; Danilenko, V N

    2018-03-10

    The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays

  4. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

    Science.gov (United States)

    Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

    1997-07-01

    Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

  5. Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs.

    Science.gov (United States)

    Bours, V; Villalobos, J; Burd, P R; Kelly, K; Siebenlist, U

    1990-11-01

    We have cloned and characterized a mitogen-inducible gene isolated from human T cells that predicts a protein of 968 amino acids. The amino-terminal domain has regions homologous to the oncogene rel and to the developmentally important gene dorsal of Drosophila. The carboxy-terminal domain contains repeat structures found in a variety of proteins that are involved in cell-cycle control of yeast and in tissue differentiation in Drosophila and Ceanorhabditis elegans, as well as in the putative human oncogene bcl-3 and in the ankyrin protein. A truncated form of the product of this gene translated in vitro is a DNA-binding protein which interacts specifically with the kappa B binding site found in many inducible genes, including the enhancer in human immunodeficiency virus. This gene is yet another in a growing list of important regulatory molecules whose expression is transcriptionally induced upon cellular activation.

  6. A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata.

    Science.gov (United States)

    Accorsi, Alice; Benatti, Stefania; Ross, Eric; Nasi, Milena; Malagoli, Davide

    2017-07-01

    The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ-specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain-containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin-like protein (PlP), that we called Pc-plp and we analyzed by real-time PCR (qPCR) its expression. In control snails, highest levels of Pc-plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc-plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc-plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc-plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc-plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain-containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  8. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  9. On-line separation of native proteins by two-dimensional liquid chromatography using a single column.

    Science.gov (United States)

    Geng, Xindu; Ke, Congyu; Chen, Gang; Liu, Peng; Wang, Fei; Zhang, Huiqiang; Sun, Xuan

    2009-04-17

    This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes--hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n(c,total) and total sample peak capacity n(c,total)(*) of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the "top-down" MS strategy in proteomics.

  10. Activated entomopathogenic nematode infective juveniles release lethal venom proteins.

    Directory of Open Access Journals (Sweden)

    Dihong Lu

    2017-04-01

    Full Text Available Entomopathogenic nematodes (EPNs are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs of Steinernema carpocapsae (a well-studied EPN species release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

  11. The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Roland Baumgartner

    Full Text Available Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig, an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1 and Caprin (Capr and directly interacts with and regulates the RNA-binding protein Rasputin (Rin in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.

  12. The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster.

    Science.gov (United States)

    Baumgartner, Roland; Stocker, Hugo; Hafen, Ernst

    2013-01-01

    Appropriate expression of growth-regulatory genes is essential to ensure normal animal development and to prevent diseases like cancer. Gene regulation at the levels of transcription and translational initiation mediated by the Hippo and Insulin signaling pathways and by the TORC1 complex, respectively, has been well documented. Whether translational control mediated by RNA-binding proteins contributes to the regulation of cellular growth is less clear. Here, we identify Lingerer (Lig), an UBA domain-containing protein, as growth suppressor that associates with the RNA-binding proteins Fragile X mental retardation protein 1 (FMR1) and Caprin (Capr) and directly interacts with and regulates the RNA-binding protein Rasputin (Rin) in Drosophila melanogaster. lig mutant organs overgrow due to increased proliferation, and a reporter for the JAK/STAT signaling pathway is upregulated in a lig mutant situation. rin, Capr or FMR1 in combination as double mutants, but not the respective single mutants, display lig like phenotypes, implicating a redundant function of Rin, Capr and FMR1 in growth control in epithelial tissues. Thus, Lig regulates cell proliferation during development in concert with Rin, Capr and FMR1.

  13. PI(4,5)P2-binding effector proteins for vesicle exocytosis

    Science.gov (United States)

    Martin, Thomas F. J.

    2014-01-01

    PI(4,5)P2 participates directly in priming and possibly fusion steps of Ca2+-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2 reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2 directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2 effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2 in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. PMID:25280637

  14. KirrelL, a member of the Ig-domain superfamily of adhesion proteins, is essential for fusion of primary mesenchyme cells in the sea urchin embryo.

    Science.gov (United States)

    Ettensohn, Charles A; Dey, Debleena

    2017-01-15

    In the sea urchin embryo, primary mesenchyme cells (PMCs) adhere to one another and fuse via filopodia, forming cable-like structures within which skeletal rods are deposited. Although this process was first described more than a century ago, molecules that participate in PMC adhesion and fusion have not been identified. Here we show that KirrelL, a PMC-specific, Ig domain-containing transmembrane protein, is essential for PMC fusion, probably by mediating filopodial adhesions that are a pre-requisite for subsequent membrane fusion. We show that KirrelL is not required for PMC specification, migration, or for direct filopodial contacts between PMCs. In the absence of KirrelL, however, filopodial contacts do not result in fusion. kirrelL is a member of a family of closely related, intronless genes that likely arose through an echinoid-specific gene expansion, possibly via retrotransposition. Our findings are significant in that they establish a direct linkage between the transcriptional network deployed in the PMC lineage and an effector molecule required for a critically important PMC morphogenetic process. In addition, our results point to a conserved role for Ig domain-containing adhesion proteins in facilitating cell fusion in both muscle and non-muscle cell lineages during animal development. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Protein Extractability

    African Journals Online (AJOL)

    Results showed that protein extractability was dependent on pH, type of salt, salt concentrations and extraction time. Salts extracted more proteins from the moringa seed flour than water. Maximum extraction of protein was. 85.06% and 84.72% with 0.5 M CaCl and 0.75 M NaCl respectively. On varying the pH, maximum ...

  16. Proteomic screen in the simple metazoan Hydra identifies 14-3-3 binding proteins implicated in cellular metabolism, cytoskeletal organisation and Ca2+ signalling

    Directory of Open Access Journals (Sweden)

    Imhof Axel

    2007-07-01

    Full Text Available Abstract Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra.

  17. Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron

    International Nuclear Information System (INIS)

    Xu, Qingping; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; 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; Lam, Winnie W.; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Yeh, Andrew; Zhou, Jiadong; 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 a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed. Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT-3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment

  18. NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

    Directory of Open Access Journals (Sweden)

    Sonnhammer Erik LL

    2006-03-01

    Full Text Available Abstract Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families.

  19. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

  20. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  1. Whey Protein

    Science.gov (United States)

    ... Fraction de Lactosérum, Fraction de Petit-Lait, Goat Milk Whey, Goat Whey, Isolat de Protéine de Lactosérum, Isolat de Protéine de Petit-Lait, Lactosérum de Lait de Chèvre, MBP, Milk Protein, Milk Protein Isolate, Mineral Whey Concentrate, Proteínas ...

  2. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  3. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at ... GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column.

  4. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  5. Molecular cloning and subcellular localization of Tektin2-binding protein 1 (Ccdc 172) in rat spermatozoa.

    Science.gov (United States)

    Yamaguchi, Airi; Kaneko, Takane; Inai, Tetsuichiro; Iida, Hiroshi

    2014-04-01

    Tektins (TEKTs) are composed of a family of filament-forming proteins localized in cilia and flagella. Five types of mammalian TEKTs have been reported, all of which have been verified to be present in sperm flagella. TEKT2, which is indispensable for sperm structure, mobility, and fertilization, was present at the periphery of the outer dense fiber (ODF) in the sperm flagella. By yeast two-hybrid screening, we intended to isolate flagellar proteins that could interact with TEKT2, which resulted in the isolation of novel two genes from the mouse testis library, referred as a TEKT2-binding protein 1 (TEKT2BP1) and -protein 2 (TEKT2BP2). In this study, we characterized TEKT2BP1, which is registered as a coiled-coil domain-containing protein 172 (Ccdc172) in the latest database. RT-PCR analysis indicated that TEKT2BP1 was predominantly expressed in rat testis and that its expression was increased after 3 weeks of postnatal development. Immunocytochemical studies discovered that TEKT2BP1 localized in the middle piece of rat spermatozoa, predominantly concentrated at the mitochondria sheath of the flagella. We hypothesize that the TEKT2-TEKT2BP1 complex might be involved in the structural linkage between the ODF and mitochondria in the middle piece of the sperm flagella.

  6. A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

    Science.gov (United States)

    Banerjee, Arka; Adolph, Ramona S.; Gopalakrishnapai, Jayashree; Kleinboelting, Silke; Emmerich, Christiane; Steegborn, Clemens; Visweswariah, Sandhya S.

    2015-01-01

    Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as “sinks” for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell. PMID:25802331

  7. Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin.

    Directory of Open Access Journals (Sweden)

    Madhusudan Venkatareddy

    Full Text Available Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase, Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

  8. Analysis of Protein-RNA and Protein-Peptide Interactions in Equine Infectious Anemia

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Hyung [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    Macromolecular interactions are essential for virtually all cellular functions including signal transduction processes, metabolic processes, regulation of gene expression and immune responses. This dissertation focuses on the characterization of two important macromolecular interactions involved in the relationship between Equine Infectious Anemia Virus (EIAV) and its host cell in horse: (1) the interaction between the EIAV Rev protein and its binding site, the Rev-responsive element (RRE) and (2) interactions between equine MHC class I molecules and epitope peptides derived from EIAV proteins. EIAV, one of the most divergent members of the lentivirus family, has a single-stranded RNA genome and carries several regulatory and structural proteins within its viral particle. Rev is an essential EIAV regulatory encoded protein that interacts with the viral RRE, a specific binding site in the viral mRNA. Using a combination of experimental and computational methods, the interactions between EIAV Rev and RRE were characterized in detail. EIAV Rev was shown to have a bipartite RNA binding domain contain two arginine rich motifs (ARMs). The RRE secondary structure was determined and specific structural motifs that act as cis-regulatory elements for EIAV Rev-RRE interaction were identified. Interestingly, a structural motif located in the high affinity Rev binding site is well conserved in several diverse lentiviral genoes, including HIV-1. Macromolecular interactions involved in the immune response of the horse to EIAV infection were investigated by analyzing complexes between MHC class I proteins and epitope peptides derived from EIAV Rev, Env and Gag proteins. Computational modeling results provided a mechanistic explanation for the experimental finding that a single amino acid change in the peptide binding domain of the quine MHC class I molecule differentially affectes the recognitino of specific epitopes by EIAV-specific CTL. Together, the findings in this

  9. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware...

  10. LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

    Science.gov (United States)

    James, Victoria; Zhang, Yining; Foxler, Daniel E; de Moor, Cornelia H; Kong, Yi Wen; Webb, Thomas M; Self, Tim J; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M; Morley, Simon J; Longmore, Gregory D; Bushell, Martin; Sharp, Tyson V

    2010-07-13

    In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.

  11. Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.

    Science.gov (United States)

    Loew, Caroline; Knoblich, Constanze; Fichtl, Jürgen; Alt, Nadja; Diepold, Katharina; Bulau, Patrick; Goldbach, Pierre; Adler, Michael; Mahler, Hanns-Christian; Grauschopf, Ulla

    2012-11-01

    The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development. Copyright © 2012 Wiley Periodicals, Inc.

  12. Crystal structure of an eIF4G-like protein from Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Euiyoung; Bitto, Eduard; Bingman, Craig A.; McCoy, Jason G.; Wesenberg, Gary E.; Phillips, Jr., George N. (UW)

    2012-04-18

    The gene LOC 91917 Danio rerio (zebrafish) encodes a protein annotated in the UniProt knowledgebase as the middle domain of eukaryotic initiation factor 4G domain containing protein b (MIF4Gdb). Its molecular weight is 25.8 kDa, and it comprises 222 amino acid residues. BLAST searches revealed homologues of D. rerio MIF4Gdb in many eukaryotes including humans. The homologue sand MIF4Gdb were identified as members of the Pfam family, MIF4G (PF2854), which is named after the middle domain of eukaryotic initiation factor 4G (eIF4G). eIF4G is a component of eukaryotic translational initiation complex, and contains binding sites for other initiation factors, suggesting its critical role in translational initiation. The MIF4G domain also occurs in several other proteins involved in RNA metabolism, including the Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), and the nuclear cap-binding protein 80-kDa subunit (CBP80). Sequence and structure analysis of the MIF4G domains in many proteins indicate that the domain assumes an all helical fold and has tandem repeated motifs. The zebrafish protein described here has homology to domains of other proteins variously referred to as NIC-containing proteins (NMD2, eIF4G, CBP80). The biological function of D. rerio MIF4Gdb has not yet been experimentally characterized, and the annotation is based on amino acid sequence comparison. D. rerio MIF4Gdb did not share more than 25% sequence identity with any protein for which the three-dimensional structure is known and was selected as a target for structure determination by the Center for Eukaryotic Structural Genomics (CESG). Here, they report the crystal structure of D. rerio MIF4Gdb (UniGene code Dr.79360, UniProt code Q5EAQ1, CESG target number GO.79294).

  13. Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

    Directory of Open Access Journals (Sweden)

    Gunjan Arora

    2014-11-01

    Full Text Available Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK PknQ.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c, a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c, which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

  14. Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

    Science.gov (United States)

    Arora, Gunjan; Sajid, Andaleeb; Singhal, Anshika; Joshi, Jayadev; Virmani, Richa; Gupta, Meetu; Verma, Nupur; Maji, Abhijit; Misra, Richa; Baronian, Grégory; Pandey, Amit K; Molle, Virginie; Singh, Yogendra

    2014-11-01

    Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

  15. A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression.

    Directory of Open Access Journals (Sweden)

    Quitterie Venot

    Full Text Available ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1 domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL, which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.

  16. Protein deamidation

    OpenAIRE

    Robinson, Noah E.

    2002-01-01

    A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically ...

  17. Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

    Science.gov (United States)

    Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

    2014-07-01

    Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

  18. The Primary Role of Fibrinogen-Related Proteins in Invertebrates Is Defense, Not Coagulation

    Science.gov (United States)

    Hanington, Patrick C.; Zhang, Si-Ming

    2010-01-01

    In vertebrates, the conversion of fibrinogen into fibrin is an essential process that underlies the establishment of the supporting protein framework required for coagulation. In invertebrates, fibrinogen-domain-containing proteins play a role in the defense response generated against pathogens; however, they do not function in coagulation, suggesting that this role has been recently acquired. Molecules containing fibrinogen motifs have been identified in numerous invertebrate organisms, and most of these molecules known to date have been linked to defense. Moreover, recent genome projects of invertebrate animals have revealed surprisingly high numbers of fibrinogen-like loci in their genomes, suggesting important and perhaps diverse functions of fibrinogen-like proteins in invertebrates. The ancestral role of molecules containing fibrinogen-related domains (FReDs) with immunity is the focus of this review, with emphasis on specific FReDs called fibrinogen-related proteins (FREPs) identified from the schistosome-transmitting mollusc Biomphalaria glabrata. Herein, we outline the range of invertebrate organisms FREPs can be found in, and detail the roles these molecules play in defense and protection against infection. PMID:21063081

  19. Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing.

    Science.gov (United States)

    Elaswad, Ahmed; Khalil, Karim; Cline, David; Page-McCaw, Patrick; Chen, Wenbiao; Michel, Maximilian; Cone, Roger; Dunham, Rex

    2018-01-20

    The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

  20. Structural basis for antagonizing a host restriction factor by C7 family of poxvirus host-range proteins.

    Science.gov (United States)

    Meng, Xiangzhi; Krumm, Brian; Li, Yongchao; Deng, Junpeng; Xiang, Yan

    2015-12-01

    Human sterile alpha motif domain-containing 9 (SAMD9) protein is a host restriction factor for poxviruses, but it can be overcome by some poxvirus host-range proteins that share homology with vaccinia virus C7 protein. To understand the mechanism of action for this important family of host-range factors, we determined the crystal structures of C7 and myxoma virus M64, a C7 family member that is unable to antagonize SAMD9. Despite their different functions and only 23% sequence identity, the two proteins have very similar overall structures, displaying a previously unidentified fold comprised of a compact 12-stranded antiparallel β-sandwich wrapped in two short α helices. Extensive structure-guided mutagenesis of C7 identified three loops clustered on one edge of the β sandwich as critical for viral replication and binding with SAMD9. The loops are characterized with functionally important negatively charged, positively charged, and hydrophobic residues, respectively, together forming a unique "three-fingered molecular claw." The key residues of the claw are not conserved in two C7 family members that do not antagonize SAMD9 but are conserved in distantly related C7 family members from four poxvirus genera that infect diverse mammalian species. Indeed, we found that all in the latter group of proteins bind SAMD9. Taken together, our data indicate that diverse mammalian poxviruses use a conserved molecular claw in a C7-like protein to target SAMD9 and overcome host restriction.

  1. A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome.

    Directory of Open Access Journals (Sweden)

    Felipe H Santiago-Tirado

    2015-05-01

    Full Text Available Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter's opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT, one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.

  2. Influence of Skyrme-type interaction on HICs observables

    Directory of Open Access Journals (Sweden)

    Zhang Yingxun

    2016-01-01

    Full Text Available A new version of the improved quantum molecular dynamics model has been developed by including Skyrme type momentum dependent interaction. 12 Skyrme like parameter sets {K0, S0, L, m*s, m*v} are adopted in the transport model code to calculate the isospin diffusion, single and double ratios of transverse emitted nucleons, neutron proton isoscaling ratios. The calculations and correlation analysis evidence that isospin diffusion observable at lower beam energy is sensitive to the slope of symmetry energy and m*s. The high energy neutrons and protons yield ratios from reactions at different incident energies provide a sensitive observable to study the nucleon effective mass splitting, at higher beam energy.

  3. The transcriptional repressor HIC1 regulates intestinal immune homeostasis

    Czech Academy of Sciences Publication Activity Database

    Burrows, K.; Antignano, F.; Bramhall, M.; Chenery, A.; Scheer, S.; Kořínek, Vladimír; Underhill, T. M.; Zaph, C.

    2017-01-01

    Roč. 10, č. 6 (2017), s. 1518-1528 ISSN 1933-0219 Institutional support: RVO:68378050 Keywords : cd4(+) t-cells * anti-interleukin-17 monoclonal-antibody * cd103(+) dendritic cells * acid receptor-alpha * retinoic acid * t(h)17 cells * target genes * double-blind * tgf-beta * differentiation Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 7.478, year: 2016

  4. Protein Crystallizability.

    Science.gov (United States)

    Smialowski, Pawel; Wong, Philip

    2016-01-01

    Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

  5. Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Ishii Marina

    2002-04-01

    Full Text Available Abstract Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside, express the green fluorescent protein (gfpuv during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1 of transformed (pGFP Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm were sonicated in successive intervals of sonication (25 vibrations/pulse to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations. The intracellular permeate with gfpuv in extraction buffer (TE solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF was subjected to the three-phase partitioning (TPP method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0. Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA, after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP

  6. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    Directory of Open Access Journals (Sweden)

    Jerry eCurran

    2015-02-01

    Full Text Available The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs, as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

  7. The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase and Other Eukaryotic BAM/GIDE Domain Proteins.

    Directory of Open Access Journals (Sweden)

    Jeremy G Wideman

    Full Text Available MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1 is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings

  8. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  9. Evaluating the performance of different multicolumn setups for chromatographic separation of proteins on hydrophobic interaction chromatography media by a numerical study.

    Science.gov (United States)

    Bochenek, Roman; Marek, Wojciech; Piątkowski, Wojciech; Antos, Dorota

    2013-08-02

    A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system. As a case study separation of a ternary mixture of proteins on HIC media has been selected. Two separation problems have been analyzed referring to the situation when the target component was the most strongly adsorbed as well as when it exhibited intermediate adsorption strength. A mathematical model has been used to simulate the process dynamics and to optimize operating conditions for the separation. The numerical study indicated that batch column arrangements can outperform SMB-based configurations regarding all performance indicators considered, which has been attributed to solvent mixing in the recycled streams and distortion of the gradient shape in SMB units. It has been concluded that the performance of complex multicolumn systems should be verified vs. batch column operations prior to the realization of the separation process. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. The nuclear matrix protein HNRNPU maintains 3D genome architecture globally in mouse hepatocytes.

    Science.gov (United States)

    Fan, Hui; Lv, Pin; Huo, Xiangru; Wu, Jicheng; Wang, Qianfeng; Cheng, Lu; Liu, Yun; Tang, Qi-Qun; Zhang, Ling; Zhang, Feng; Zheng, Xiaoqi; Wu, Hao; Wen, Bo

    2018-02-01

    Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization. © 2018 Fan et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    International Nuclear Information System (INIS)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João

    2013-01-01

    cytoskeleton and cell structure (paramyosin), while exposure to Ag + had a strong influence in one protein related to stress response (putative c1q domain containing protein) and two proteins involved in cytoskeleton and cell structure (actin and α-tubulin). Protein identification showed that Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades (including mitochondria and nucleus) that can lead to cell death. This toxicity represents the cumulative effect of Ag + released from the particles and other properties as particle size and surface reactivity. This study helped to unravel the molecular mechanisms that can be associated with Ag NPs toxicity; nevertheless, some additional studies are required to investigate the exact interaction between these NPs and cellular components

  12. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João, E-mail: mbebian@ualg.pt

    2013-07-15

    protein involved in cytoskeleton and cell structure (paramyosin), while exposure to Ag{sup +} had a strong influence in one protein related to stress response (putative c1q domain containing protein) and two proteins involved in cytoskeleton and cell structure (actin and α-tubulin). Protein identification showed that Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades (including mitochondria and nucleus) that can lead to cell death. This toxicity represents the cumulative effect of Ag{sup +} released from the particles and other properties as particle size and surface reactivity. This study helped to unravel the molecular mechanisms that can be associated with Ag NPs toxicity; nevertheless, some additional studies are required to investigate the exact interaction between these NPs and cellular components.

  13. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation

    Energy Technology Data Exchange (ETDEWEB)

    LeNoue-Newton, Michele; Watkins, Guy R.; Zou, Ping; Germane, Katherine L.; McCorvey, Lisa R.; Wadzinski, Brian E.; Spiller, Benjamin W. (Vanderbilt)

    2012-04-30

    Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: (1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and (2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

  14. Recombinant protein production technology

    Science.gov (United States)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  15. Efficient expression of codon-adapted affinity tagged super folder green fluorescent protein for synchronous protein localization and affinity purification studies in Tetrahymena thermophila.

    Science.gov (United States)

    Yilmaz, Gürkan; Arslanyolu, Muhittin

    2015-03-25

    A superior Green Fluorescent Protein (GFP) mutant, known as superfolder GFP (sfGFP), is more soluble, faster folding, and is the brightest of the known GFP mutants. This study aimed to create a codon-adapted sfGFP tag (TtsfGFP) for simultaneous protein localization and affinity purification in Tetrahymena thermophila. In vivo fluorescence spectroscopic analyses of clones carrying a codon-adapted and 6 × His tagged TtsfGFP cassette showed approximately 2-4-fold increased fluorescence emission compared with the control groups at 3 h. Fluorescence microscopy also revealed that TtsfGFP reached its emission maxima at 100 min, which was much earlier than controls expressing EGFP and sfGFP (240 min). A T. thermophila ATP-dependent DNA ligase domain containing hypothetical gene (H) was cloned into the 3' end of 6 × His-TtsfGFP to assess the affinity/localization dual tag feature. Fluorescence microscopy of the 6 × His-TtsfGFP-H clone confirmed its localization in the macro- and micronucleus of vegetative T. thermophila. Simultaneous affinity purification of TtsfGFP and TtsfGFP-H with Ni-NTA beads was feasible, as shown by Ni-NTA purified proteins analysis by SDS-PAGE and western blotting. We successfully codon adapted the N-terminal 6 × His-TtsfGFP tag and showed that it could be used for protein localization and affinity purification simultaneously in T. thermophila. We believe that this dual tag will advance T. thermophila studies by providing strong visual traceability of the target protein in vivo and in vitro during recombinant production of heterologous and homologous proteins.

  16. The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing.

    Science.gov (United States)

    Vogl, Christian; Butola, Tanvi; Haag, Natja; Hausrat, Torben J; Leitner, Michael G; Moutschen, Michel; Lefèbvre, Philippe P; Speckmann, Carsten; Garrett, Lillian; Becker, Lore; Fuchs, Helmut; Hrabe de Angelis, Martin; Nietzsche, Sandor; Kessels, Michael M; Oliver, Dominik; Kneussel, Matthias; Kilimann, Manfred W; Strenzke, Nicola

    2017-11-01

    Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model. © 2017 The Authors.

  17. Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o.

    Science.gov (United States)

    Jeon, Jae-Pyo; Thakur, Dhananjay P; Tian, Jin-Bin; So, Insuk; Zhu, Michael X

    2016-05-15

    Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  18. NMR structure of the mouse prion protein domain PrP(121-231).

    Science.gov (United States)

    Riek, R; Hornemann, S; Wider, G; Billeter, M; Glockshuber, R; Wüthrich, K

    1996-07-11

    The 'protein only' hypothesis states that a modified form of normal prion protein triggers infectious neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), or Creutzfeldt-Jakob disease (CJD) in humans. Prion proteins are thought to exist in two different conformations: the 'benign' PrPcform, and the infectious 'scrapie form', PrPsc. Knowledge of the three-dimensional structure of PrPc is essential for understanding the transition to PrPsc. The nuclear magnetic resonance (NMR) structure of the autonomously folding PrP domain comprising residues 121-231 (ref. 6) contains a two-stranded antiparallel beta-sheet and three alpha-helices. This domain contains most of the point-mutation sites that have been linked, in human PrP, to the occurrence of familial prion diseases. The NMR structure shows that these mutations occur within, or directly adjacent to, regular secondary structures. The presence of a beta-sheet in PrP(121-231) is in contrast with model predictions of an all-helical structure of PrPc (ref. 8), and may be important for the initiation of the transition from PrPc to PrPsc.

  19. Cell cycle dependent association of EBP50 with protein phosphatase 2A in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Anita Boratkó

    Full Text Available Ezrin-radixin-moesin (ERM-binding phosphoprotein 50 (EBP50 is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC. Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A. Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

  20. Aquaporin Protein-Protein Interactions

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    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  1. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  2. Structural basis and kinetics of force-induced conformational changes of an αA domain-containing integrin.

    Directory of Open Access Journals (Sweden)

    Xue Xiang

    Full Text Available Integrin α(Lβ₂ (lymphocyte function-associated antigen, LFA-1 bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1 when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α₇-helix, have been suggested to correspond to three different affinity states for ligand binding.The kinetics of the force-driven transitions between these conformations has not been defined and dynamically coupled to the force-dependent dissociation from ligand. Here we show, by steered molecular dynamics (SMD simulations, that the αA domain was successively transitioned through three distinct conformations upon pulling the C-terminus of its α₇-helix. Based on these sequential transitions, we have constructed a mathematical model to describe the coupling between the αA domain conformational changes of LFA-1 and its dissociation from ICAM-1 under force. Using this model to analyze the published data on the force-induced dissociation of single LFA-1/ICAM-1 bonds, we estimated the force-dependent kinetic rates of interstate transition from the short-lived to intermediate-lived and from intermediate-lived to long-lived states. Interestingly, force increased these transition rates; hence activation of LFA-1 was accelerated by pulling it via an engaged ICAM-1.Our study defines the structural basis for mechanical regulation of the kinetics of LFA-1 αA domain conformational changes and relates these simulation results to experimental data of force-induced dissociation of single LFA-1/ICAM-1 bonds by a new mathematical model, thus provided detailed structural and kinetic characterizations for force-stabilization of LFA-1/ICAM-1 interaction.

  3. CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens

    Czech Academy of Sciences Publication Activity Database

    von Schwartzenberg, K.; Lindner, A.C.; Gruhn, N.; Šimura, Jan; Novák, Ondřej; Strnad, Miroslav; Gonneau, M.; Nogue, F.; Heyl, A.

    2016-01-01

    Roč. 67, č. 3 (2016), s. 667-679 ISSN 0022-0957 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : cytokinin * Bryophyte * cytokinin receptor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.830, year: 2016

  4. Ankyrin repeat and zinc-finger domain-containing 1 mutations are associated with infantile-onset inflammatory bowel disease

    NARCIS (Netherlands)

    Van Haaften-Visser, Désirée Y.; Harakalova, Magdalena; Mocholi, Enric; Van Montfrans, Joris M.; Elkadri, Abdul; Rieter, Ester; Fiedler, Karoline; Van Hasselt, Peter M.; Triffaux, Emily M.M.; Van Haelst, Mieke M.; Nijman, Isaac J.; Kloosterman, Wigard P.; Nieuwenhuis, Edward E S; Muise, Aleixo M.; Cuppen, Edwin; Houwen, Roderick H.J.; Coffer, Paul J.

    2017-01-01

    Infantile-onset inflammatory bowel disease (IO IBD) is an invalidating illness with an onset before 2 years of age and has a complex pathophysiology in which genetic factors are important. Homozygosity mapping and whole-exome sequencing in an IO IBD patient and subsequent sequencing of the candidate

  5. DMPD: TIR domain-containing adaptors define the specificity of TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available e (.html) CSML File (.csml) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  6. Virus-Induced Chaperone-Enriched (VICE domains function as nuclear protein quality control centers during HSV-1 infection.

    Directory of Open Access Journals (Sweden)

    Christine M Livingston

    2009-10-01

    Full Text Available Virus-Induced Chaperone-Enriched (VICE domains form adjacent to nuclear viral replication compartments (RC during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70, the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42 degrees C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.

  7. Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo

    Directory of Open Access Journals (Sweden)

    Christine Tucher

    2018-03-01

    Full Text Available Extracellular vesicles (EVs are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs released from the cellular surface, while exosomes are small EVs (SEVs released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction. We could clearly separate two EV populations, namely SEVs (average diameter <200 nm and LEVs (diameter range between 200 and 1000 nm. Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70 or tumor susceptibility 101 (TSG101, microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3 turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10 as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with

  8. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.

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    Michael Brauchle

    Full Text Available Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.

  9. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    Science.gov (United States)

    Becherelli, Marco; Prachi, Prachi; Viciani, Elisa; Biagini, Massimiliano; Fiaschi, Luigi; Chiarot, Emiliano; Nosari, Sarah; Brettoni, Cecilia; Marchi, Sara; Biancucci, Marco; Fontana, Maria Rita; Montagnani, Francesca; Bagnoli, Fabio; Barocchi, Michèle A; Manetti, Andrea G O

    2013-01-01

    Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  10. Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

    Directory of Open Access Journals (Sweden)

    Marco Becherelli

    Full Text Available Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

  11. Disruption of a Ciliary B9 Protein Complex Causes Meckel Syndrome

    Science.gov (United States)

    Dowdle, William E.; Robinson, Jon F.; Kneist, Andreas; Sirerol-Piquer, M. Salomé; Frints, Suzanna G.M.; Corbit, Kevin C.; Zaghloul, Norran A.; van Lijnschoten, Gesina; Mulders, Leon; Verver, Dideke E.; Zerres, Klaus; Reed, Randall R.; Attié-Bitach, Tania; Johnson, Colin A.; García-Verdugo, José Manuel; Katsanis, Nicholas; Bergmann, Carsten; Reiter, Jeremy F.

    2011-01-01

    Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS. PMID:21763481

  12. Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

    Directory of Open Access Journals (Sweden)

    Inna A. Abdeeva

    2018-04-01

    Full Text Available The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57 in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1 and putative protein phosphatase (At2g30020; АТ2, whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1 and RmlC-like cupin (At5g39120. The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1 and clathrin adaptor complex-related protein (At5g05010. Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

  13. AcEST: DK955695 [AcEST

    Lifescience Database Archive (English)

    Full Text Available l repressor NF-X1 OS=Mus musc... 29 9.3 sp|Q9JLZ6|HIC2_MOUSE Hypermethylated in cancer 2 protein OS=Mus ... ...TGSLIEQLTTEKYECMVCCELVQVTAPVWS 367 >sp|Q9JLZ6|HIC2_MOUSE Hypermethylated in cancer 2 protein OS=Mus musculus

  14. Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1

    Science.gov (United States)

    Reinecke, James B.; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2014-01-01

    ABSTRACT Localization of the non-receptor tyrosine kinase Src to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and the movement of Src to the plasma membrane is mediated by endocytic transport. However, the precise pathways and regulatory proteins that are responsible for SRC transport are incompletely understood. Here, we demonstrate that Src partially colocalizes with the endocytic regulatory protein MICAL-L1 (molecule interacting with CasL-like protein 1) in mammalian cells. Furthermore, MICAL-L1 is required for growth-factor- and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner EHD1 (EH domain-containing protein 1) is also required for Src activation and transport. Moreover, the MICAL-L1-mediated recruitment of EHD1 to Src-containing recycling endosomes is required for the release of Src from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mechanism by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases. PMID:24481818

  15. Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

    Science.gov (United States)

    Shih, Ying-Chun; Chen, Chao-Ling; Zhang, Yan; Mellor, Rebecca L; Kanter, Evelyn M; Fang, Yun; Wang, Hua-Chi; Hung, Chen-Ting; Nong, Jing-Yi; Chen, Hui-Ju; Lee, Tzu-Han; Tseng, Yi-Shuan; Chen, Chiung-Nien; Wu, Chau-Chung; Lin, Shuei-Liong; Yamada, Kathryn A; Nerbonne, Jeanne M; Yang, Kai-Chien

    2018-03-13

    Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure (HF). Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting co-expression gene network analysis on RNA sequencing data from failing human heart, we identified thioredoxin domain containing 5 (TXNDC5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum (ER) protein, as a potential novel mediator of cardiac fibrosis and we completed experiments to test this hypothesis directly. Objective: To determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. Methods and Results: RNASeq and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles (LV) and in hypertrophied/failing mouse LV. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 (TGFβ1) and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under TGFβ1 stimulation in vitro. Knockdown of TXNDC5 attenuated TGFβ1-induced hCF activation and ECM protein upregulation independent of SMAD3, whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing JNK activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. TGFβ1-induced TXNDC5 upregulation in hCF was dependent on ER stress and

  16. Preliminary study on plasma proteins in pregnant and non-pregnant female dogs.

    Science.gov (United States)

    Szczubiał, Marek; Wawrzykowski, Jacek; Dąbrowski, Roman; Krawczyk, Magdalena; Kankofer, Marta

    2017-07-15

    In this study, we used a combined approach based on 2-dimensional electrophoresis (2-DE), difference in gel electrophoresis (DIGE), and mass spectrometry (MS) to identify the plasma protein composition in pregnant female dogs and compared it with non-pregnant female dogs. We used the plasma samples obtained from four female dogs during I, II, and III thirds of pregnancy, three days after parturition, as well as from four non-pregnant female dogs in diestrus phase. Analysis of 2-DE gel image exhibited of 249 protein spots. The intensity of staining of 35 spots differed significantly (P dogs. We used matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI TOF MS) to identify 47 spots corresponding to 52 different proteins. Five identified protein spots, including zinc finger BED domain-containing protein 5, hemoglobin subunit beta-2, integrator complex subunit 7, apolipoprotein A-I, and glutamyl aminopeptidase were differentially presented in the plasma of pregnant and non-pregnant female dogs. To the best of our knowledge, this is the first report on the plasma protein profile of pregnant and non-pregnant female dogs. In this study, we identified proteins that have not been previously identified in dogs. Our findings showed that numerous protein spots were differentially presented in the plasma of female dogs during normal pregnancy. Although we identified only a limited number of differentially presented proteins, our study demonstrated that the plasma protein profile changed during pregnancy in female dogs, which suggests its importance in maintaining pregnancy. Further studies are necessary to define complete plasma protein profile of pregnant female dogs and to identify all proteins that are differentially presented in the pregnant animals compared with the non-pregnant ones. In addition, studies are warranted to explain the role of those proteins in maintaining the pregnancy and their usefulness in detection of early pregnancy

  17. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

    Science.gov (United States)

    Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

    2012-11-26

    In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  18. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

    Directory of Open Access Journals (Sweden)

    Petitjean Céline

    2012-11-01

    Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  19. The skeletal proteome of the brittle star Ophiothrix spiculata identifies C-type lectins and other proteins conserved in echinoderm skeleton formation

    Directory of Open Access Journals (Sweden)

    Brian T. Livingston

    2016-07-01

    Full Text Available Determining the identity and functional role of proteins involved in biomineralization and the formation of skeletons is critical to our understanding of the process. Proteomics has allowed rapid characterization of the proteins occluded within mineralized tissue, but the large numbers of proteins detected makes it difficult to assign the relative importance of each protein. We have taken a comparative approach, examining the skeletal proteome of different species of echinoderms in order to identify the proteins that are conserved and likely to be important. Our previous study comparing the skeletal proteome of the brittle star Ophiocoma wendtii to the published proteomes of the sea urchin Strongylocentrotus purpuratus revealed some conservation of proteins, but indicated that the C-type lectin domain-containing spicule matrix proteins abundant in the sea urchin skeletal proteome were not conserved in the brittle star. Here we examine the skeletal proteome of a different species of brittle star, Ophiothrix spiculata. We have isolated the proteins from the skeleton of O. spiculata and performed LC/MS/MS to identify peptides present. Comparison to transcriptome and genome databases revealed the proteins present in the O. spiculata proteome. Despite being diverged for several million years, the two brittle stars have very similar proteins in their skeletons. Included is a fibrinogen C-like lectin and several C-type lectins proteins, which we describe in detail. The unusual number of C-type lectins found in the S. purpuatus skeleton and the repetitive regions seen in those spicule matrix proteins are not present in O. spiculata.

  20. Learning about Proteins

    Science.gov (United States)

    ... Videos for Educators Search English Español Learning About Proteins KidsHealth / For Kids / Learning About Proteins What's in ... from the foods you eat. Different Kinds of Protein Protein from animal sources, such as meat and ...

  1. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development.

    Science.gov (United States)

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D; McComb, Rodney D; Muirhead, David; Todd, Gordon L; Gould, Karen; Datta, Kaustubh; Gelineau-van Waes, Janee; Band, Vimla; Band, Hamid

    2016-02-17

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

  2. Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.

    Science.gov (United States)

    Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T; Chazin, Walter J; Kiyokawa, Hiroaki; Yin, Jun

    2018-01-01

    E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

  3. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Mate Suzanne E

    2012-09-01

    Full Text Available Abstract Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ. We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

  4. Modulation of matrix mineralization by Vwc2-like protein and its novel splicing isoforms.

    Science.gov (United States)

    Ohyama, Yoshio; Katafuchi, Michitsuna; Almehmadi, Ahmed; Venkitapathi, Sundharamani; Jaha, Haytham; Ehrenman, Jason; Morcos, Joseph; Aljamaan, Reem; Mochida, Yoshiyuki

    2012-02-03

    In search of new cysteine knot protein (CKP) family members, we found a novel gene called von Willebrand factor C domain-containing protein 2-like (Vwc2l, also known as Brorin-like) and its transcript variants (Vwc2l-1, Vwc2l-2 and Vwc2l-3). Based on the deduced amino acid sequence, Vwc2l-1 has a signal peptide and 2 cysteine-rich (CR) domains, while Vwc2l-2 lacks a part of 2nd CR domain and Vwc2l-3 both CR domains. Although it has been reported that the expression of Brorin-like was predominantly observed in brain, we found that Vwc2l transcript variants were detected in more ubiquitous tissues. In osteoblasts, the induction of Vwc2l expression was observed at matrix mineralization stage. When Vwc2l was stably transfected into osteoblasts, the matrix mineralization was markedly accelerated in Vwc2l-expressing clones compared to that in the control, indicating the modulatory effect of Vwc2l protein on osteoblastic cell function. The mechanistic insight of Vwc2l-modulation was further investigated and we found that the expression of Osterix, one of the key osteogenic markers, was significantly increased by addition of all Vwc2l isoform proteins. Taken together, Vwc2l is a novel secreted protein that promotes matrix mineralization by modulating Osterix expression likely through TGF-β superfamily growth factor signaling pathway. Our data may provide mechanistic insights into the biological functions of this novel CKP member in bone and further suggest a novel approach to enhance osteoblast function, which enables to accerelate bone formation, regeneration and healing. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Fusion-protein-assisted protein crystallization.

    Science.gov (United States)

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  6. Membrane bending by protein-protein crowding.

    Science.gov (United States)

    Stachowiak, Jeanne C; Schmid, Eva M; Ryan, Christopher J; Ann, Hyoung Sook; Sasaki, Darryl Y; Sherman, Michael B; Geissler, Phillip L; Fletcher, Daniel A; Hayden, Carl C

    2012-09-01

    Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.

  7. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  8. Backbone and side-chain 1H, 13C and 15N assignments of the ubiquitin-associated domain of human X-linked inhibitor of apoptosis protein

    Science.gov (United States)

    Hui, Sin-Kam; Tse, Man-Kit; Yang, Yinhua; Wong, Benjamin Chun-Yu

    2009-01-01

    X-linked inhibitor of apoptosis protein (XIAP), a leading member of the family of inhibitor of apoptosis (IAP) proteins, is considered as the most potent and versatile inhibitor of caspases and apoptosis. It has been reported that XIAP is frequently overexpressed in cancer and its expression level is implicated in contributing to tumorigenesis, disease progression, chemoresistance and poor patient-survival. Therefore, XIAP is one of the leading targets in drug development for cancer therapy. Recently, based on bioinformatics study, a previously unrecognized but evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs was identified. The UBA domain is found to be essential for the oncogenic potential of IAP, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP to activate NF-κB. In the present study, we report the near complete resonance assignments of the UBA domain-containing region of human XIAP protein. Secondary structure prediction based on chemical shift index (CSI) analysis reveals that the protein is predominately α-helical, which is consistent with the structures of known UBA proteins. PMID:19916060

  9. AcEST: BP920653 [AcEST

    Lifescience Database Archive (English)

    Full Text Available chnera aphi... 31 3.1 sp|Q9UJ42|GP160_HUMAN Probable G-protein coupled receptor 160 OS... 30 6.8 sp|Q9R1Y5|HIC1_MOUSE Hyperme...thylated in cancer 1 protein OS=Mus ... 29 8.7 sp|Q14526|HIC1_HUMAN Hypermethylated in canc... Query: 376 AAHC 387 + HC Sbjct: 171 SRHC 174 >sp|Q9R1Y5|HIC1_MOUSE Hypermethylated in cancer 1 protein OS=M...SL 850 >sp|Q14526|HIC1_HUMAN Hypermethylated in cancer 1 protein OS=Homo sapiens GN=HIC1 PE=1 SV=4 Length =

  10. System-wide characterization of bZIP transcription factor proteins involved in infection-related morphogenesis of Magnaporthe oryzae.

    Science.gov (United States)

    Tang, Wei; Ru, Yanyan; Hong, Li; Zhu, Qian; Zuo, Rongfang; Guo, Xianxian; Wang, Jingzhen; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2015-04-01

    The basic leucine zipper (bZIP) domain-containing transcription factors (TFs) function as key regulators of cellular growth and differentiation in eukaryotic organisms including fungi. We have previously identified MoAp1 and MoAtf1 as bZIP TFs in Magnaporthe oryzae and demonstrated that they regulate the oxidative stress response and are critical in conidiogenesis and pathogenicity. Studies of bZIP proteins could provide a novel strategy for controlling rice blast, but a systematic examination of the bZIP proteins has not been carried out. Here, we identified 19 additional bZIP TFs and characterized their functions. We found that the majority of these TFs exhibit active functions, most notably, in conidiogenesis. We showed that MoHac1 regulates the endoplasmic reticulum stress response through a conserved unfolded protein response pathway, MoMetR controls amino acid metabolism to govern growth and differentiation, and MoBzip10 governs appressorium function and invasive hyphal growth. Moreover, MoBzip5 participates in appressorium formation through a pathway distinct from that MoBzip10, and MoMeaB appears to exert a regulatory role through nutrient uptake and nitrogen utilization. Collectively, our results provide insights into shared and specific functions associated with each of these TFs and link the regulatory roles to the fungal growth, conidiation, appressorium formation, host penetration and pathogenicity. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    Science.gov (United States)

    Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

  12. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    Directory of Open Access Journals (Sweden)

    Nan Zhang

    Full Text Available Zea mays (maize Opaque-2 (ZmO2 protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2 as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

  13. The muscle regulatory and structural protein MLP is a cytoskeletal binding partner of betaI-spectrin.

    Science.gov (United States)

    Flick, M J; Konieczny, S F

    2000-05-01

    Muscle LIM protein (MLP) is a striated muscle-specific factor that enhances myogenic differentiation and is critical to maintaining the structural integrity of the contractile apparatus. The ability of MLP to regulate myogenesis is particularly interesting since it exhibits multiple subcellular localizations, being found in both nuclear and cytoplasmic compartments. Despite extensive biochemical analyses on MLP, the mechanism(s) by which it influences the myogenic program remains largely undefined. To further examine the role of MLP as a positive myogenic regulator, a yeast two-hybrid screen was employed to identify cytoplasmic-associated MLP binding partners. From this screen, the cytoskeletal protein betaI-spectrin was isolated. Protein interaction assays demonstrate that MLP and betaI-spectrin associate with one another in vivo as well as when tested under several in vitro binding conditions. betaI-spectrin binds specifically to MLP but not to the MLP related proteins CRP1 and CRP2 or to other LIM domain containing proteins. The MLP:beta-spectrin interaction is mediated by the second LIM motif of MLP and by repeat 7 of beta-spectrin. Confocal microscopy studies also reveal that MLP co-localizes with beta-spectrin at the sarcolemma overlying the Z- and M-lines of myofibrils in both cardiac and skeletal muscle tissue. Given that beta-spectrin is a known costamere protein, we propose that sarcolemma-associated MLP also serves as a key costamere protein, stabilizing the association of the contractile apparatus with the sarcolemma by linking the beta-spectrin network to the alpha-actinin crosslinked actin filaments of the myofibril.

  14. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  15. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  16. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  17. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  18. Ontological visualization of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hill David P

    2005-02-01

    Full Text Available Abstract Background Cellular processes require the interaction of many proteins across several cellular compartments. Determining the collective network of such interactions is an important aspect of understanding the role and regulation of individual proteins. The Gene Ontology (GO is used by model organism databases and other bioinformatics resources to provide functional annotation of proteins. The annotation process provides a mechanism to document the binding of one protein with another. We have constructed protein interaction networks for mouse proteins utilizing the information encoded in the GO annotations. The work reported here presents a methodology for integrating and visualizing information on protein-protein interactions. Results GO annotation at Mouse Genome Informatics (MGI captures 1318 curated, documented interactions. These include 129 binary interactions and 125 interaction involving three or more gene products. Three networks involve over 30 partners, the largest involving 109 proteins. Several tools are available at MGI to visualize and analyze these data. Conclusions Curators at the MGI database annotate protein-protein interaction data from experimental reports from the literature. Integration of these data with the other types of data curated at MGI places protein binding data into the larger context of mouse biology and facilitates the generation of new biological hypotheses based on physical interactions among gene products.

  19. 24-hour urine protein

    Science.gov (United States)

    Urine protein - 24 hour; Chronic kidney disease - urine protein; Kidney failure - urine protein ... Heart failure High blood pressure during pregnancy ( preeclampsia ) Kidney disease caused by diabetes, high blood pressure, autoimmune disorders, ...

  20. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  1. Protein in diet

    Science.gov (United States)

    Diet - protein ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a ... to eat animal products to get all the protein you need in your diet. Amino acids are ...

  2. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  3. Structure, evolution, and expression of antimicrobial silk proteins, seroins in Lepidoptera.

    Science.gov (United States)

    Dong, Zhaoming; Song, Qianru; Zhang, Yan; Chen, Shiyi; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

    2016-08-01

    The silks of silkworm and waxworm contain abundant antimicrobial proteins, including protease inhibitors and seroins. Protease inhibitors have antifungal activities, whereas seroins have antiviral and antibacterial activities. In order to obtain insights into the structure, evolution, and expression of seroins, we performed an extensive survey based on the available genome, transcriptome, and expressed sequence tags datasets. Sixty-four seroins were identified in 32 lepidopteran species. The phylogenetic and structural analyses revealed that seroins can be classified into five subfamilies: seroin 1, seroin 2, seroin 3, seroin 2 + 1, and seroin 3 + 3. It is interesting that seroin 2 + 1 contains two tandem seroin domains, seroin 2 and seroin 1, whereas seroin 3 + 3 has two tandem seroin 3 domains. Each seroin domain contains a proline-rich N-terminal motif and a conserved C-terminal motif. The transcriptome and EST data indicated that seroin 1 and seroin 2 genes were expressed in the silk gland but seroin 3 genes were not. Semi-quantitative RT-PCR and western blot analyses suggested that seroin 1 and seroin 2 were constantly accumulated in the silk gland of silkworm during the fifth instar, and then secreted into cocoon silk during spinning. Immunofluorescence analyses indicated that seroin 1 was secreted into the fibroin and sericin layers, whereas seroin 2 protein was only secreted into the sericin layer. However, the antimicrobial activity of seroin 2 was more effective than that of seroin 1. The presence of seroin 1 in the fibroin layer suggested that this protein not only acts as an antimicrobial protein, but might also play a role in the assembly and secretion of fibroins. Seroin 3, which was first identified here, might be related to pheromone synthesis or recognition, as it was highly expressed in male antennae and in the pheromone gland. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Monoliths from poly(ethylene glycol) diacrylate and dimethacrylate for capillary hydrophobic interaction chromatography of proteins.

    Science.gov (United States)

    Li, Yuanyuan; Tolley, H Dennis; Lee, Milton L

    2010-07-23

    Rigid monoliths were synthesized solely from poly(ethylene glycol) diacrylates (PEGDA) or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths by one-step UV-initiated polymerization. Methanol/ethyl ether and cyclohexanol/decanol were used as bi-porogen mixtures for the PEGDA and PEGDMA monoliths, respectively. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. For tri- and tetra-ethylene glycol diacrylates (i.e., PEGDA 258 and PEGDA 302), most combinations of methanol and ethyl ether were effective in forming monoliths, while for diacrylates containing longer chain lengths (i.e., PEGDA 575 and PEGDA 700), polymerization became more sensitive to the bi-porogen ratio. A similar tendency was also observed for PEGDMA monomers. Polymerization of monoliths was conducted at approximately 0 degrees C and room temperature, which produced significant differences in monolith morphology and permeability. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution in hydrophobic interaction chromatography (HIC). Detailed study of these monoliths demonstrated that chromatographic performance was not affected by changing the ratios of the two porogens, but resulted in almost identical retention times and comparable peak capacities. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. Mass recoveries for test proteins were found to be greater than 90, indicating its excellent biocompatibility. All monoliths demonstrated nearly no swelling or shrinking in different polarity solvents, and most of them could be stored dry, indicating excellent stability due to their highly crosslinked networks. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The

  5. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  6. Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

    Science.gov (United States)

    Nagel, Marie-Kristin; Kalinowska, Kamila; Vogel, Karin; Reynolds, Gregory D; Wu, Zhixiang; Anzenberger, Franziska; Ichikawa, Mie; Tsutsumi, Chie; Sato, Masa H; Kuster, Bernhard; Bednarek, Sebastian Y; Isono, Erika

    2017-08-22

    Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta , indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

  7. A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies.

    Directory of Open Access Journals (Sweden)

    Cécile Fourrage

    Full Text Available Poc1 (Protein of Centriole 1 proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

  8. Nanotechnologies in protein microarrays.

    Science.gov (United States)

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures.

  9. Interference of HTLV-1 Tax Protein with Cell Polarity Regulators: Defining the Subcellular Localization of the Tax-DLG1 Interaction.

    Science.gov (United States)

    Marziali, Federico; Bugnon Valdano, Marina; Brunet Avalos, Clarisse; Moriena, Lucía; Cavatorta, Ana Laura; Gardiol, Daniela

    2017-11-23

    Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain-containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions.

  10. FBW7 loss promotes epithelial-to-mesenchymal transition in non-small cell lung cancer through the stabilization of Snail protein.

    Science.gov (United States)

    Zhang, Yong; Zhang, Xinxin; Ye, Mingxiang; Jing, Pengyu; Xiong, Jie; Han, Zhiping; Kong, Jing; Li, Mengyang; Lai, Xiaofeng; Chang, Ning; Zhang, Jian; Zhang, Jian

    2018-04-10

    The E3 ubiquitin ligase F-box and WD repeat domain containing 7 (FBW7α) functions as a putative tumor suppressor in non-small cell lung cancer (NSCLC) due to its regulation of a set of oncogenic proteins associated with cell proliferation and mitosis. Increasing efforts have been focused on the understanding of FBW7 in determining cell cycle progression and apoptosis induction, however, the correlation between FBW7 and tumor metastasis is not fully understood. In this study, we reported a potential anti-metastatic effect of FBW7 in non-small cell lung cancer (NSCLC). In this model, FBW7 inhibited cancer cell metastasis primarily by inducing ubiquitination and proteolysis of the transcriptional factor Snail, which suppressed E-cadherin cell tight junction protein expression. Loss of FBW7 would stabilize the Snail protein, thus, inhibit E-cadherin expression and promote metastasis in vitro and in vivo. Moreover, Snail ubiquitination and degradation were also achieved by pharmacological approach, in which the FBW7 agonist oridonin treatment led to Snail proteolysis. Furthermore, FBW7 silencing stabilized Snail protein and induced epithelial-to mesenchymal transition (EMT), and acquisition of migration and invasion properties in NSCLC. Overall, our study provides new insights into the FBW7-Snail axis in regulating cell migration and invasion, and suggests that targeting FBW7 may be a potent approach to inhibit metastasis in NSCLC. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  11. Physical, Functional and Genetic Interactions between the BEACH Domain Protein SPIRRIG and LIP5 and SKD1 and Its Role in Endosomal Trafficking to the Vacuole in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Alexandra Steffens

    2017-11-01

    Full Text Available Beige and Chediak Higashi (BEACH domain-containing proteins (BDCPs are facilitators of membrane-dependent cellular processes in eukaryotes. Mutations in BDCPs cause malfunctions of endosomal compartments in various cell types. Recently, the molecular analysis of the BDCP homolog gene SPIRRIG (SPI has revealed a molecular function in P-bodies and the regulation of RNA stability. We therefore aimed to analyze, whether SPI has also a role in membrane-dependent processes. In this study, we show that SPI physically interacts with endosomal sorting complex required for transport associated ATPase Suppressor of K+-transport growth defect1 (SKD1 and its positive regulator, LYST Interacting Protein 5 (LIP5 and report genetic interactions between SPI and SKD1 and LIP5. We further show that the endosomal transport route of soluble proteins to the lytic vacuole is disturbed in spi lip5 double mutants but not in the single mutants. These vacuolar transport defects were suppressed by additional expression of SKD1. Our results indicate that the BEACH domain protein SPI has in addition to a role in P-bodies a function in endosomal transport routes.

  12. Structure of human stabilin-1 interacting chitinase-like protein (SI-CLP) reveals a saccharide-binding cleft with lower sugar-binding selectivity.

    Science.gov (United States)

    Meng, Geng; Zhao, Yanmei; Bai, Xiaoyun; Liu, Yong; Green, Todd J; Luo, Ming; Zheng, Xiaofeng

    2010-12-17

    Human secreted protein stabilin-1 interacting chitinase-like protein (SI-CLP) has been identified as a novel member of Glyco_18 domain-containing proteins that is involved in host defense and inflammatory reactions. Efficient secretion of SI-CLP is mediated by its interaction with the endocytic/sorting receptor stabilin-1. SI-CLP is expressed abundantly in macrophages and neutrophils and is up-regulated by Th2 cytokine IL-4 and glucocorticoid, which suggest that SI-CLP could be a marker for adverse effects of glucocorticoid therapy. To gain insight into the biological function of SI-CLP, we determined the crystal structure of SI-CLP at 2.7 Å resolution by x-ray crystallography and found that it featured a typical triose-phosphate isomerase barrel fold with a putative saccharide-binding cleft. Comparison with other chitinase-like proteins showed the cleft to be atypically wide and open. The saccharide-binding capacity of SI-CLP was investigated, and its ligand-binding specificity was found to relate to the length of the oligosaccharides, with preference for chitotetraose. Further investigations reveal that SI-CLP could bind LPS in vitro and neutralize its endotoxin effect on macrophages. Our results demonstrate the saccharide-binding property of SI-CLP by structure and in vitro biochemical analyses and suggest the possible roles of SI-CLP in pathogen sensing and endotoxin neutralization.

  13. The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

    Directory of Open Access Journals (Sweden)

    Alexandra Steffens

    2015-07-01

    Full Text Available Members of the highly conserved class of BEACH domain containing proteins (BDCPs have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1, associates to mRNA processing bodies (P-bodies, and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

  14. Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

    Science.gov (United States)

    Kanda, Yuki; Satoh, Ryosuke; Matsumoto, Saki; Ikeda, Chisato; Inutsuka, Natsumi; Hagihara, Kanako; Matzno, Sumio; Tsujimoto, Sho; Kita, Ayako; Sugiura, Reiko

    2016-08-15

    The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process. © 2016. Published by The Company of Biologists Ltd.

  15. PsHint1, associated with the G-protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae.

    Science.gov (United States)

    Zhang, Xin; Zhai, Chunhua; Hua, Chenlei; Qiu, Min; Hao, Yujuan; Nie, Pingping; Ye, Wenwu; Wang, Yuanchao

    2016-02-01

    Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G-protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1-interacting proteins, including PsHint1, a histidine triad (HIT) domain-containing protein orthologous to human HIT nucleotide-binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms of PsGPA1. An analysis of the gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα-independent pathway involved in the pathogenicity of P. sojae. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  16. Neuronal RING finger protein 11 (RNF11 regulates canonical NF-κB signaling

    Directory of Open Access Journals (Sweden)

    Pranski Elaine L

    2012-04-01

    Full Text Available Abstract Background The RING domain-containing protein RING finger protein 11 (RNF11 is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. Methods and results Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11’s association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and

  17. Antibody-validated proteins in inflamed islets of fulminant type 1 diabetes profiled by laser-capture microdissection followed by mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Yoriko Nishida

    Full Text Available There are no reports of proteomic analyses of inflamed islets in type 1 diabetes.Proteins expressed in the islets of enterovirus-associated fulminant type 1 diabetes (FT1DM with extensive insulitis were identified by laser-capture microdissection mass spectrometry using formalin-fixed paraffin-embedded pancreatic tissues.Thirty-eight proteins were identified solely in FT1DM islets, most of which have not been previously linked to type 1 diabetes. Five protein-protein interacting clusters were identified, and the cellular localization of selected proteins was validated immunohistochemically. Migratory activity-related proteins, including plastin-2 (LCP1, moesin (MSN, lamin-B1 (LMNB1, Ras GTPase-activating-like protein (IQGAP1 and others, were identified in CD8+ T cells and CD68+ macrophages infiltrated to inflamed FT1DM islets. Proteins involved in successive signaling in innate/adaptive immunity were identified, including SAM domain and HD domain-containing protein 1 (SAMHD1, Ras GTPase-activating-like protein (IQGAP1, proteasome activator complex subunit 1 (PSME1, HLA class I histocompatibility antigen (HLA-C, and signal transducer and activator of transcription 1-alpha/beta (STAT1. Angiogenic (thymidine phosphorylase (TYMP and anti-angiogenic (tryptophan-tRNA ligase (WARS factors were identified in migrating CD8+ T cells and CD68+ macrophages. Proteins related to virus replication and cell proliferation, including probable ATP-dependent RNA helicase DEAD box helicase 5 (DDX5 and heterogeneous nuclear ribonucleoprotein H (HNRNPH1, were identified. The anti-apoptotic protein T-complex protein 1 subunit epsilon (CCT5, the anti-oxidative enzyme 6-phosphogluconate dehydrogenase (PDG, and the anti-viral and anti-apoptotic proteins serpin B6 (SERPINB6 and heat shock 70 kDa protein1-like (HSPA1L, were identified in FT1DM-affected islet cells.The identified FT1DM-characterizing proteins include those involved in aggressive beta cell destruction through

  18. Backbone and side-chain ¹H, ¹³C and ¹⁵N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii.

    Science.gov (United States)

    Tse, Man-Kit; Cheung, Stanley K K; Ke, Yi-hong; Lau, Candy C Y; Sze, Kong-Hung; Yuen, Kwok-Yung

    2014-04-01

    Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

  19. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  20. The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

    Science.gov (United States)

    Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

    2017-10-01

    A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. An Arabidopsis jmjC domain protein protects transcribed genes from DNA methylation at CHG sites.

    Science.gov (United States)

    Miura, Asuka; Nakamura, Miyuki; Inagaki, Soichi; Kobayashi, Akie; Saze, Hidetoshi; Kakutani, Tetsuji

    2009-04-22

    Differential cytosine methylation of genes and transposons is important for maintaining integrity of plant genomes. In Arabidopsis, transposons are heavily methylated at both CG and non-CG sites, whereas the non-CG methylation is rarely found in active genes. Our previous genetic analysis suggested that a jmjC domain-containing protein IBM1 (increase in BONSAI methylation 1) prevents ectopic deposition of non-CG methylation, and this process is necessary for normal Arabidopsis development. Here, we directly determined the genomic targets of IBM1 through high-resolution genome-wide analysis of DNA methylation. The ibm1 mutation induced extensive hyper-methylation in thousands of genes. Transposons were unaffected. Notably, long transcribed genes were most severely affected. Methylation of genes is limited to CG sites in wild type, but CHG sites were also methylated in the ibm1 mutant. The ibm1-induced hyper-methylation did not depend on previously characterized components of the RNAi-based DNA methylation machinery. Our results suggest novel transcription-coupled mechanisms to direct genic methylation not only at CG but also at CHG sites. IBM1 prevents the CHG methylation in genes, but not in transposons.

  2. Protein digestion in ruminants

    African Journals Online (AJOL)

    digestibility, or the contribution of endogenous protein to the indigestible feed .... endogenous protein fractions. Alternatively, Stern & Satter (1984) suggested a method whereby the increased protein outflow to the small intestine, resulting from the incremental addition of ..... definition of the various protein fractions. Finally ...

  3. The Protein Model Portal

    OpenAIRE

    Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

    2008-01-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

  4. Photoswitchable cyan fluorescent protein for protein tracking.

    Science.gov (United States)

    Chudakov, Dmitriy M; Verkhusha, Vladislav V; Staroverov, Dmitry B; Souslova, Ekaterina A; Lukyanov, Sergey; Lukyanov, Konstantin A

    2004-11-01

    In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.

  5. Neto1 is a novel CUB-domain NMDA receptor-interacting protein required for synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    David Ng

    2009-02-01

    Full Text Available The N-methyl-D-aspartate receptor (NMDAR, a major excitatory ligand-gated ion channel in the central nervous system (CNS, is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1, a complement C1r/C1s, Uegf, Bmp1 (CUB domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.

  6. ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production.

    Directory of Open Access Journals (Sweden)

    Gabrielle Vieyres

    2016-04-01

    Full Text Available Hepatitis C virus (HCV particles closely mimic human very-low-density lipoproteins (VLDL to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58 as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

  7. Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding.

    Directory of Open Access Journals (Sweden)

    Qianting Zhai

    Full Text Available Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC(Gag protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements.We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding.Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  8. Zinc finger protein 521 overexpression increased transcript levels of ...

    Indian Academy of Sciences (India)

    2016-02-12

    Feb 12, 2016 ... Dulbecco's modified Eagle medium/Ham's-Nutrient Mixture F-12; EB, embryoid body; EBF1, early B-cell factor 1; FITC, fluorescein isothiocyanate;. Fndc5, firbonectin type III domain containing 5; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; LIF, leukemia inhibitory factor; mESCs, mouse embryonic ...

  9. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  10. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

    Energy Technology Data Exchange (ETDEWEB)

    Heintz, Udo; Meinhart, Anton; Winkler, Andreas, E-mail: andreas.winkler@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Heidelberg (Germany)

    2014-03-01

    Crystal structures of two truncated variants of the transcription factor PpsR from R. sphaeroides are presented that enabled the phasing of a triple PAS domain construct. Together, these structures reveal the importance of α-helical PAS extensions for multi-PAS domain-mediated protein oligomerization and function. Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR from Rhodobacter sphaeroides is such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsR{sub Q-PAS1}) and two (PpsR{sub N-Q-PAS1}) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR{sub ΔHTH}) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR{sub ΔHTH} structure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that

  11. Crystal Structure of VC0702 at 2.0 Angstrom: Conserved Hypothetical Protein from Vibrio Cholerae

    Energy Technology Data Exchange (ETDEWEB)

    Ni,S.; Forouhar, F.; Bussiere, D.; Robinson, H.; Kennedy, M.

    2006-01-01

    VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 Angstroms and refined to R{sub work} = 22.8% and R{sub free} = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a = 66.61 Angstroms, b = 88.118 Angstroms, and c = 118.35 Angstroms with a homodimer in the asymmetric unit. VC0702, which forms a mixed {alpha} + {beta} three-layered {alpha}{beta}{alpha} sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae.

  12. Crystal Structure of VC0702 at 2.0 Angstrom: Conserved Hypothetical Protein from Vibrio Cholerae

    International Nuclear Information System (INIS)

    Ni, S.; Forouhar, F.; Bussiere, D.; Robinson, H.; Kennedy, M.

    2006-01-01

    VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 Angstroms and refined to R work = 22.8% and R free = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a = 66.61 Angstroms, b = 88.118 Angstroms, and c = 118.35 Angstroms with a homodimer in the asymmetric unit. VC0702, which forms a mixed α + β three-layered αβα sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae

  13. Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

    Science.gov (United States)

    Rust, K; Grosso, L; Zhang, V; Chang, D; Persson, A; Longmore, W; Cai, G Z; Crouch, E

    1991-10-01

    Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

  14. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

    Science.gov (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

    2018-05-01

    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  15. Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

    Science.gov (United States)

    Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

    2015-04-20

    During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity.

    Science.gov (United States)

    Caira, Simonetta; Iannelli, Antonio; Sciarrillo, Rosaria; Picariello, Gianluca; Renzone, Giovanni; Scaloni, Andrea; Addeo, Pietro

    2017-12-01

    The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and β-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

  17. Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly

    Directory of Open Access Journals (Sweden)

    Martin Kornecki

    2017-09-01

    Full Text Available Significant progress in the manufacturing of biopharmaceuticals has been made by increasing the overall titers in the USP (upstream processing titers without raising the cost of the USP. In addition, the development of platform processes led to a higher process robustness. Despite or even due to those achievements, novel challenges are in sight. The higher upstream titers created more complex impurity profiles, both in mass and composition, demanding higher separation capacities and selectivity in downstream processing (DSP. This creates a major shift of costs from USP to DSP. In order to solve this issue, USP and DSP integration approaches can be developed and used for overall process optimization. This study focuses on the characterization and classification of host cell proteins (HCPs in each unit operation of the DSP (i.e., aqueous two-phase extraction, integrated countercurrent chromatography. The results create a data-driven feedback to the USP, which will serve for media and process optimizations in order to reduce, or even eliminate nascent critical HCPs. This will improve separation efficiency and may lead to a quantitative process understanding. Different HCP species were classified by stringent criteria with regard to DSP separation parameters into “The Good, the Bad, and the Ugly” in terms of pI and MW using 2D-PAGE analysis depending on their positions on the gels. Those spots were identified using LC-MS/MS analysis. HCPs, which are especially difficult to remove and persistent throughout the DSP (i.e., “Bad” or “Ugly”, have to be evaluated by their ability to be separated. In this approach, HCPs, considered “Ugly,” represent proteins with a MW larger than 15 kDa and a pI between 7.30 and 9.30. “Bad” HCPs can likewise be classified using MW (>15 kDa and pI (4.75–7.30 and 9.30–10.00. HCPs with a MW smaller than 15 kDa and a pI lower than 4.75 and higher than 10.00 are classified as “Good” since their

  18. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, whereas vertebrates contain two to four genes. In cnidarians, the gene appears to encode a secreted protein, but transmembrane isoforms of the protein have also evolved, and in many species, alternative splicing facilitates the expression of both transmembrane and secreted isoforms. In most species......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...

  19. Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

    Science.gov (United States)

    McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

    2017-07-11

    The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the

  20. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Peng Liu

    2015-01-01

    Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

  1. Personalizing Protein Nourishment

    Science.gov (United States)

    DALLAS, DAVID C.; SANCTUARY, MEGAN R.; QU, YUNYAO; KHAJAVI, SHABNAM HAGHIGHAT; VAN ZANDT, ALEXANDRIA E.; DYANDRA, MELISSA; FRESE, STEVEN A.; BARILE, DANIELA; GERMAN, J. BRUCE

    2016-01-01

    Proteins are not equally digestible—their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources and processing methods must be tailored to the consumer’s digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health. PMID:26713355

  2. Extração, purificação e caracterização físico-química da proteína verde fluorescente recombinante (GFPuv expressa em Escherichia coli Extraction, purification and physical-chemical characterization of recombinant green fluorescent protein (GFPuv expressed in Escherichia coli cells

    Directory of Open Access Journals (Sweden)

    Eb Chiarini

    2003-12-01

    Full Text Available O aumento do uso da proteína verde fluorescente (GFPuv como ferramenta de pesquisas biotecnológicas requer um estudo mais cuidadoso das propriedades bioquímicas e físicas da molécula de GFPuv. Este trabalho teve como objetivo a aplicação de métodos físicos e químicos para o isolamento, a extração da GFPuv de células de Escherichia coli DH5-±, purificação da proteína, e o estudo da estabilidade desta em diferentes valores de pH. Células de E. coli expressando GFPuv foram submetidas a quatro ciclos sucessivos de congelamento e descongelamento intercalados por sonicação (CDS, para promover a permeação seletiva da GFPuv. Os permeados foram submetidos à extração por partição em três fases (TPP e posterior purificação por eluição da proteína em coluna cromatográfica de interação hidrofóbica (HIC.Obteve-se rendimento semelhante em GFPuv no 1º ciclo de permeação seletiva (CDS e por extração (TPP associada à purificação (HIC para os quais impurezas não foram visualizadas por eletroforese. As estruturas moleculares da GFPuv extraída e purificada mostraram-se inalteradas em valores de pH entre 6,0 e 9,8, e foram confirmadas nos espectros de emissão e de excitação.The recombinant green fluorescent protein (GFPuv has been used as a marker in several research fields. The purpose of the present work was to evaluate the influence of the selective physical permeation procedure applied to the cells of Escherichia coli for the extraction of GFPuv in relation to the chemical procedures of extraction and purification. Transformed cells (0.92-1.44 mg/mL of E. coli DH5-a expressing GFPuv were submitted to four cycles (1º, 2º, 3º, 4º of freezing (-20 ºC/ 0.83 ºC/ min/thawing interlaid by sonication (3 pulses/6 s/25 vibrations. The intracellular permeate with GFPuv in buffer solution (Tris-HCl 25 mM pH 8.0 + b-mercaptoethanol (1 mM + PMSF (0.1 mM was submitted to the three-phase partitioning (TPP method and

  3. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  4. The Three-Dimensional Solution Structure of the Src Homology Domain-2 of the Growth Factor Receptor-Bound Protein-2

    International Nuclear Information System (INIS)

    Senior, Mary M.; Frederick, Anne F.; Black, Stuart; Murgolo, Nicholas J.; Perkins, Louise M.; Wilson, Oswald; Snow, Mark E.; Wang Yusen

    1998-01-01

    A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central β-sheet. The core structure of the SH2 domain contains an antiparallel β-sheet flanked by two parallel α-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions

  5. Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

    Science.gov (United States)

    Guo, Emily Z; Xu, Zhaohui

    2015-03-27

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

    Science.gov (United States)

    Guo, Emily Z.; Xu, Zhaohui

    2015-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

  7. Revisiting the Phylogeny of the Animal Formins: Two New Subtypes, Relationships with Multiple Wing Hairs Proteins, and a Lost Human Formin.

    Directory of Open Access Journals (Sweden)

    David Pruyne

    Full Text Available Formins are a widespread family of eukaryotic cytoskeleton-organizing proteins. Many species encode multiple formin isoforms, and for animals, much of this reflects the presence of multiple conserved subtypes. Earlier phylogenetic analyses identified seven major formin subtypes in animals (DAAM, DIAPH, FHOD, FMN, FMNL, INF, and GRID2IP/delphilin, but left a handful of formins, particularly from nematodes, unassigned. In this new analysis drawing from genomic data from a wider range of taxa, nine formin subtypes are identified that encompass all the animal formins analyzed here. Included in this analysis are Multiple Wing Hairs proteins (MWH, which bear homology to formin N-terminal domains. Originally identified in Drosophila melanogaster and other arthropods, MWH-related proteins are also identified here in some nematodes (including Caenorhabditis elegans, and are shown to be related to a novel MWH-related formin (MWHF subtype. One surprising result of this work is the discovery that a family of pleckstrin homology domain-containing formins (PHCFs is represented in many vertebrates, but is strikingly absent from placental mammals. Consistent with a relatively recent loss of this formin, the human genome retains fragments of a defunct homologous formin gene.

  8. Products of the unc-52 gene in Caenorhabditis elegans are homologous to the core protein of the mammalian basement membrane heparan sulfate proteoglycan.

    Science.gov (United States)

    Rogalski, T M; Williams, B D; Mullen, G P; Moerman, D G

    1993-08-01

    Mutations in the unc-52 gene of Caenorhabditis elegans affect attachment of the myofilament lattice to the muscle cell membrane. Here, we demonstrate that the unc-52 gene encodes a nematode homolog of perlecan, the mammalian basement membrane heparan sulfate proteoglycan. The longest potential open reading frame of this gene encodes a 2482-amino-acid protein with a signal peptide and four domains. The first domain is unique to the unc-52 polypeptide, whereas the three remaining domains contain sequences found in the LDL receptor (domain II) laminin (domain III) and N-CAM (domain IV). We have identified three alternatively spliced transcripts that encode different carboxy-terminal sequences. The two larger transcripts encode proteins containing all or part of domain IV, whereas the smaller transcript encodes a shortened polypeptide that completely lacks domain IV. We have determined that the disorganized muscle phenotype observed in unc-52(st196) animals is caused by the insertion of a Tc1 transposon into domain IV. Two monoclonal antibodies that recognize an extracellular component of all contractile tissues in C. elegans fail to stain embryos homozygous for a lethal unc-52 allele. We have mapped the epitopes recognized by both monoclonal antibodies to a region of domain IV in the unc-52-encoded protein sequence.

  9. Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Pedreño, María A

    2014-04-01

    Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  10. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  11. Protein electrophoresis - urine

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003589.htm Urine protein electrophoresis test To use the sharing features on this page, please enable JavaScript. The urine protein electrophoresis (UPEP) test is used to estimate how much ...

  12. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  13. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  14. Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

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    Evaristus Chibunna Mbanefo

    Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

  15. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Science.gov (United States)

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  16. Mutations in BIN1 associated with centronuclear myopathy disrupt membrane remodeling by affecting protein density and oligomerization.

    Directory of Open Access Journals (Sweden)

    Tingting Wu

    Full Text Available The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM, where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found 'budding' structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.

  17. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Directory of Open Access Journals (Sweden)

    Luís Korrodi-Gregório

    2013-03-01

    Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs. In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4 that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier.

  18. Protein sequence databases.

    Science.gov (United States)

    Apweiler, Rolf; Bairoch, Amos; Wu, Cathy H

    2004-02-01

    A variety of protein sequence databases exist, ranging from simple sequence repositories, which store data with little or no manual intervention in the creation of the records, to expertly curated universal databases that cover all species and in which the original sequence data are enhanced by the manual addition of further information in each sequence record. As the focus of researchers moves from the genome to the proteins encoded by it, these databases will play an even more important role as central comprehensive resources of protein information. Several the leading protein sequence databases are discussed here, with special emphasis on the databases now provided by the Universal Protein Knowledgebase (UniProt) consortium.

  19. Protein hydration and dynamics

    International Nuclear Information System (INIS)

    Nakagawa, Hiroshi; Kataoka, Mikio

    2015-01-01

    Inelastic neutron scattering can measure the protein thermal fluctuations under the physiological aqueous environment, especially it is powerful to observe the low-energy protein dynamics in THz region, which are revealed theoretically to be coupled with solvations. Neutron enables the selective observation of protein and hydration water by deuteration