Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

NARCIS (Netherlands)

Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

2009-01-01

Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

osFP: a web server for predicting the oligomeric states of fluorescent proteins.

Science.gov (United States)

Simeon, Saw; Shoombuatong, Watshara; Anuwongcharoen, Nuttapat; Preeyanon, Likit; Prachayasittikul, Virapong; Wikberg, Jarl E S; Nantasenamat, Chanin

2016-01-01

Currently, monomeric fluorescent proteins (FP) are ideal markers for protein tagging. The prediction of oligomeric states is helpful for enhancing live biomedical imaging. Computational prediction of FP oligomeric states can accelerate the effort of protein engineering efforts of creating monomeric FPs. To the best of our knowledge, this study represents the first computational model for predicting and analyzing FP oligomerization directly from the amino acid sequence. After data curation, an exhaustive data set consisting of 397 non-redundant FP oligomeric states was compiled from the literature. Results from benchmarking of the protein descriptors revealed that the model built with amino acid composition descriptors was the top performing model with accuracy, sensitivity and specificity in excess of 80% and MCC greater than 0.6 for all three data subsets (e.g. training, tenfold cross-validation and external sets). The model provided insights on the important residues governing the oligomerization of FP. To maximize the benefit of the generated predictive model, it was implemented as a web server under the R programming environment. osFP affords a user-friendly interface that can be used to predict the oligomeric state of FP using the protein sequence. The advantage of osFP is that it is platform-independent meaning that it can be accessed via a web browser on any operating system and device. osFP is freely accessible at http://codes.bio/osfp/ while the source code and data set is provided on GitHub at https://github.com/chaninn/osFP/.Graphical Abstract.

Transient oligomerization of the SARS-CoV N protein--implication for virus ribonucleoprotein packaging.

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Chung-ke Chang

Full Text Available The nucleocapsid (N phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD. A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

Bacterial flagellar capping proteins adopt diverse oligomeric states

Energy Technology Data Exchange (ETDEWEB)

Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.; Yu, Xiong; Diederichs, Kay; Helmsing, Saskia; Vromen, Aviv; Friedler, Assaf; Hust, Michael; Egelman, Edward H.; Beckett, Dorothy; Wintrode, Patrick L.; Sundberg, Eric J. (UV); (Braunschweig); (Maryland-MED); (Konstanz); (Maryland); (Hebrew)

2016-09-24

Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins at the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.

Oligomeric protein structure networks: insights into protein-protein interactions

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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

Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

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Stefan Gahbauer

2016-10-01

Full Text Available The dimerization or even oligomerization of G protein coupled receptors (GPCRs causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.

Genetic noise control via protein oligomerization

Energy Technology Data Exchange (ETDEWEB)

Ghim, C; Almaas, E

2008-06-12

Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamical role of protein-protein associations. We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch), integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast protein binding-unbinding kinetics, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its intrinsic switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced) state from randomly being induced (uninduced). The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of phenotypically important toggle switches, and nested positive feedback loops in

Cartilage oligomeric matrix protein (COMP) in rheumatoid arthritis and its correlation with sonographic knee cartilage thickness and disease activity.

Science.gov (United States)

Sakthiswary, Rajalingham; Rajalingam, Shamala; Hussein, Heselynn; Sridharan, Radhika; Asrul, Abdul Wahab

2017-12-01

The aim of the study is to investigate the correlation of serum cartilage oligomeric matrix protein (COMP) levels with articular cartilage damage based on sonographic knee cartilage thickness (KCT) and disease activity in rheumatoid arthritis (RA). A total of 61 RA patients and 27 healthy controls were recruited in this study. Serum samples were obtained from all subjects to determine the serum COMP levels. All subjects had bilateral ultrasound scan of their knees. The KCT was based on the mean of measurements at three sites: the medial condyle, lateral condyle and intercondylar notch. Besides, the RA patients were assessed for their disease activity based on 28-joint-based Disease Activity Score (DAS 28). Serum COMP concentrations were significantly elevated in the RA patients compared to the controls (p = 0.001). The serum COMP levels had an inverse relationship with bilateral KCT in RA subjects and the healthy controls. COMP correlated significantly with disease activity based on DAS 28 (r = 0.299, p = 0.010), disease duration (r = 0.439, p = correlation between serum COMP and DAS 28 scores was comparable to the traditional markers of inflammation: erythrocyte sedimentation rate (ESR) (r = 0.372, p = 0.003) and C-reactive protein (CRP) (r = 0.305, p = 0.017). The serum COMP is a promising biomarker in RA which reflects disease activity and damage to the articular cartilage.

Induced oligomerization targets Golgi proteins for degradation in lysosomes.

Science.gov (United States)

Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

2015-12-01

Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Cartilage oligomeric matrix protein associates differentially with erosions and synovitis and has a different temporal course in cyclic citrullinated peptide antibody (anti-CCP)-positive versus anti-CCP-negative early rheumatoid arthritis

DEFF Research Database (Denmark)

Christensen, Anne F; Lindegaard, Hanne; Hørslev-Petersen, Kim

2011-01-01

-suppressive effect. We aimed to compare circulating cartilage oligomeric matrix protein (COMP), a marker of cartilage turnover, in untreated anti-CCP-positive and anti-CCP-negative RA, and to study the temporal pattern of COMP through 4 years of treatment, including the relationship to imaging and clinical findings.......048). In anti-CCP-positive patients, COMP exhibited a parabolic course over 4 years, while COMP in anti-CCP-negative patients had an almost linear course. In anti-CCP-positive patients, COMP was associated with MRI edema and erosion score, while COMP was correlated with synovitis score in anti...

DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.

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Jorge Mansilla-Soto

2009-07-01

Full Text Available Rep68 is a multifunctional protein of the adeno-associated virus (AAV, a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3 helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag and bovine papillomavirus (BPV E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID and an AAA(+ motor domain. The AAA(+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA(+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides

International Nuclear Information System (INIS)

Heintz, Udo; Meinhart, Anton; Winkler, Andreas

2014-01-01

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 Q-PAS1 ) and two (PpsR N-Q-PAS1 ) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsR ΔHTH ) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsR 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 plays an important

Autosomal dominant precocious osteoarthropathy due to a mutation of the cartilage oligomeric matrix protein (COMP) gene: further expansion of the phenotypic variations of COMP defects

Energy Technology Data Exchange (ETDEWEB)

Kawaji, Hiroyuki [Department of Orthopaedic Surgery, Sanyudo Hospital, 6-1-219 Chuou, Yonezawa, Yamagata 992-0045 (Japan); Nishimura, Gen [Department of Radiology, Nasu Chuou Hospital, Tochigi (Japan); Watanabe, Sobei; Sasaki, Akira; Sano, Tokuhisa [Department of Orthopaedic Surgery, Tohoku Kohsei-Nenkin Hospital, Miyagi (Japan); Mabuchi, Akihiko; Ikeda, Toshiyuki; Ikegawa, Shiro [Laboratory for Bone and Joint Diseases, SNP Research Center, Tokyo (Japan); Ohashi, Hirofumi [Division of Medical Genetics, Saitama Children' s Medical Center, Saitama (Japan)

2002-12-01

We report on a Japanese family of four generations with an autosomal dominant precocious osteoarthropathy. The cardinal clinical manifestations of affected individuals were painful weight-bearing large joints, which started in late childhood or adolescence. The radiological hallmarks included coxa plana, mild epiphyseal dysplasia of the knee, and round talar domes with tibiotalar slant in childhood, which evolved into degenerative joint diseases in adulthood. The disease phenotype was cosegregated with a mutation of the cartilage oligomeric matrix protein (COMP) gene in the family members, who underwent molecular evaluation. COMP mutations have been reported in a mild form of multiple epiphyseal dysplasia (MED), Ribbing type, as well as allied disorders with more severe manifestations, such as MED Fairbank type and pseudoachondroplasia. Unlike previously reported cases with the Ribbing type, the present patients did not have short stature or brachydactyly. This report expands further the phenotypic variations of COMP defects. (orig.)

Increased serum cartilage oligomeric matrix protein levels and decreased patellar bone mineral density in patients with chondromalacia patellae.

Science.gov (United States)

Murphy, E; FitzGerald, O; Saxne, T; Bresnihan, B

2002-11-01

Chondromalacia patellae is a potentially disabling disorder characterised by features of patellar cartilage degradation. To evaluate markers of cartilage and bone turnover in patients with chondromalacia patellae. 18 patients with chondromalacia patellae were studied. Serum cartilage oligomeric matrix protein (s-COMP) and bone sialoprotein (s-BSP) levels were measured by enzyme linked immunosorbent assay (ELISA) and compared with those of age and sex matched healthy control subjects. Periarticular bone mineral density (BMD) of both knee joints was assessed by dual energy x ray absorptiometry (DXA). s-COMP levels were significantly raised in all patients with chondromalacia patellae compared with healthy control subjects (p=0.0001). s-BSP levels did not differ significantly between the groups (p=0.41). BMD of the patella was significantly reduced in patients with chondromalacia patellae compared with the control subjects (p=0.016). In patients with bilateral chondromalacia patellae, BMD of the patella was lower in the more symptomatic knee joint (p=0.005). Changes in periarticular BMD were localised to the patella and were not present in femoral regions. Neither s-COMP (p=0.18) nor s-BSP (p=0.40) levels correlated with patellar BMD. Increased s-COMP levels, reflecting cartilage degradation, and reduced BMD localised to the patella may represent clinically useful markers in the diagnosis and monitoring of patients with chondromalacia patellae. Measures of cartilage degradation did not correlate with loss of patellar bone density, suggesting dissociated pathophysiological mechanisms.

The role of oligomerization and cooperative regulation in protein function: the case of tryptophan synthase.

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M Qaiser Fatmi

Full Text Available The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS. TRPS uses a set of α/β-dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand-bound conformations. Our simulations also revealed that the α/β-dimeric unit stabilizes the substrate-protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

Arc is a flexible modular protein capable of reversible self-oligomerization

Science.gov (United States)

Myrum, Craig; Baumann, Anne; Bustad, Helene J.; Flydal, Marte Innselset; Mariaule, Vincent; Alvira, Sara; Cuéllar, Jorge; Haavik, Jan; Soulé, Jonathan; Valpuesta, José Maria; Márquez, José Antonio; Martinez, Aurora; Bramham, Clive R.

2015-01-01

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes. PMID:25748042

Improvement of reduced serum cartilage oligomeric matrix protein levels in systemic juvenile idiopathic arthritis patients treated with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

Science.gov (United States)

Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

2009-01-01

In this study, we determined serum cartilage oligomeric matrix protein (COMP) levels in systemic juvenile idiopathic arthritis (sJIA) patients during both the active and the remission phases to investigate how the growth cartilage turnover changed under tocilizumab treatment. Specimens were collected from 201 healthy children under 16 years of age with no growth impairment, and paired sera were collected from 11 sJIA patients treated with tocilizumab. Disease activity was assessed from white blood cell count, erythrocyte sedimentation rate, C-reactive protein, and ferritin, and the COMP concentration was determined by sandwich enzyme-linked immunosorbent assay. Serum COMP concentrations were found independent of age, and the mean value in healthy children was 17.74+/-5.6 U/L. The mean serum COMP in sJIA patients during the active phase was 10.75+/-3.9 U/L, lower than that of healthy children. The mean serum COMP in the remission phase (14.89+/-3.9 U/L) was significantly higher than that in the active period (P<0.05). These results suggested that in sJIA patients, a reduced serum COMP concentration is a useful marker of active disease and growth impairment, and that the growth cartilage turnover suppressed during the active phase is improved in the remission phase under tocilizumab treatment.

Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

Science.gov (United States)

Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

2017-06-01

Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu2+ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Receptor oligomerization in family B1 of G-protein-coupled receptors

DEFF Research Database (Denmark)

Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

2012-01-01

, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...

Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

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Shekhtman Alexander

2009-04-01

Full Text Available Abstract Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

Elucidation of amyloid beta-protein oligomerization mechanisms: discrete molecular dynamics study.

Science.gov (United States)

Urbanc, B; Betnel, M; Cruz, L; Bitan, G; Teplow, D B

2010-03-31

Oligomers of amyloid beta-protein (Abeta) play a central role in the pathology of Alzheimer's disease. Of the two predominant Abeta alloforms, Abeta(1-40) and Abeta(1-42), Abeta(1-42) is more strongly implicated in the disease. We elucidated the structural characteristics of oligomers of Abeta(1-40) and Abeta(1-42) and their Arctic mutants, [E22G]Abeta(1-40) and [E22G]Abeta(1-42). We simulated oligomer formation using discrete molecular dynamics (DMD) with a four-bead protein model, backbone hydrogen bonding, and residue-specific interactions due to effective hydropathy and charge. For all four peptides under study, we derived the characteristic oligomer size distributions that were in agreement with prior experimental findings. Unlike Abeta(1-40), Abeta(1-42) had a high propensity to form paranuclei (pentameric or hexameric) structures that could self-associate into higher-order oligomers. Neither of the Arctic mutants formed higher-order oligomers, but [E22G]Abeta(1-40) formed paranuclei with a similar propensity to that of Abeta(1-42). Whereas the best agreement with the experimental data was obtained when the charged residues were modeled as solely hydrophilic, further assembly from spherical oligomers into elongated protofibrils was induced by nonzero electrostatic interactions among the charged residues. Structural analysis revealed that the C-terminal region played a dominant role in Abeta(1-42) oligomer formation whereas Abeta(1-40) oligomerization was primarily driven by intermolecular interactions among the central hydrophobic regions. The N-terminal region A2-F4 played a prominent role in Abeta(1-40) oligomerization but did not contribute to the oligomerization of Abeta(1-42) or the Arctic mutants. The oligomer structure of both Arctic peptides resembled Abeta(1-42) more than Abeta(1-40), consistent with their potentially more toxic nature.

The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

Science.gov (United States)

Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

2012-10-26

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

Genetic noise control via protein oligomerization

Directory of Open Access Journals (Sweden)

Almaas Eivind

2008-11-01

Full Text Available Abstract Background Gene expression in a cell entails random reaction events occurring over disparate time scales. Thus, molecular noise that often results in phenotypic and population-dynamic consequences sets a fundamental limit to biochemical signaling. While there have been numerous studies correlating the architecture of cellular reaction networks with noise tolerance, only a limited effort has been made to understand the dynamic role of protein-protein interactions. Results We have developed a fully stochastic model for the positive feedback control of a single gene, as well as a pair of genes (toggle switch, integrating quantitative results from previous in vivo and in vitro studies. In particular, we explicitly account for the fast binding-unbinding kinetics among proteins, RNA polymerases, and the promoter/operator sequences of DNA. We find that the overall noise-level is reduced and the frequency content of the noise is dramatically shifted to the physiologically irrelevant high-frequency regime in the presence of protein dimerization. This is independent of the choice of monomer or dimer as transcription factor and persists throughout the multiple model topologies considered. For the toggle switch, we additionally find that the presence of a protein dimer, either homodimer or heterodimer, may significantly reduce its random switching rate. Hence, the dimer promotes the robust function of bistable switches by preventing the uninduced (induced state from randomly being induced (uninduced. Conclusion The specific binding between regulatory proteins provides a buffer that may prevent the propagation of fluctuations in genetic activity. The capacity of the buffer is a non-monotonic function of association-dissociation rates. Since the protein oligomerization per se does not require extra protein components to be expressed, it provides a basis for the rapid control of intrinsic or extrinsic noise. The stabilization of regulatory circuits

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

Science.gov (United States)

Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

2017-04-14

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dimerization and oligomerization of the chaperone calreticulin

DEFF Research Database (Denmark)

Jørgensen, Charlotte S; Ryder, L Rebekka; Steinø, Anne

2003-01-01

protein. Using PAGE, urea gradient gel electrophoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5-6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 m or SDS concentrations above...... that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 m...

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

Science.gov (United States)

Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

2017-10-01

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal Structure of the Marburg Virus VP35 Oligomerization Domain

Energy Technology Data Exchange (ETDEWEB)

Bruhn, Jessica F.; Kirchdoerfer, Robert N.; Urata, Sarah M.; Li, Sheng; Tickle, Ian J.; Bricogne, Gérard; Saphire, Erica Ollmann (Scripps); (Globel Phasing); (UCSD)

2016-11-09

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (generaMarburgvirusandEbolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.

IMPORTANCEMarburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the

Free-energy landscape of protein oligomerization from atomistic simulations

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Barducci, Alessandro; Bonomi, Massimiliano; Prakash, Meher K.; Parrinello, Michele

2013-01-01

In the realm of protein–protein interactions, the assembly process of homooligomers plays a fundamental role because the majority of proteins fall into this category. A comprehensive understanding of this multistep process requires the characterization of the driving molecular interactions and the transient intermediate species. The latter are often short-lived and thus remain elusive to most experimental investigations. Molecular simulations provide a unique tool to shed light onto these complex processes complementing experimental data. Here we combine advanced sampling techniques, such as metadynamics and parallel tempering, to characterize the oligomerization landscape of fibritin foldon domain. This system is an evolutionarily optimized trimerization motif that represents an ideal model for experimental and computational mechanistic studies. Our results are fully consistent with previous experimental nuclear magnetic resonance and kinetic data, but they provide a unique insight into fibritin foldon assembly. In particular, our simulations unveil the role of nonspecific interactions and suggest that an interplay between thermodynamic bias toward native structure and residual conformational disorder may provide a kinetic advantage. PMID:24248370

An Asymmetric Deuterium Labeling Strategy to Identify Interprotomer and Intraprotomer NOEs in Oligomeric Proteins

International Nuclear Information System (INIS)

Jasanoff, Alan

1998-01-01

A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs. In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers. The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks. NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions. Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing with trypsin to yield only protomers with the desired N- and C-termini. In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present. This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments. Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates

Domain architecture and oligomerization properties of the paramyxovirus PIV 5 hemagglutinin-neuraminidase (HN) protein.

Science.gov (United States)

Yuan, Ping; Leser, George P; Demeler, Borries; Lamb, Robert A; Jardetzky, Theodore S

2008-09-01

The mechanism by which the paramyxovirus hemagglutinin-neuraminidase (HN) protein couples receptor binding to activation of virus entry remains to be fully understood, but the HN stalk is thought to play an important role in the process. We have characterized ectodomain constructs of the parainfluenza virus 5 HN to understand better the underlying architecture and oligomerization properties that may influence HN functions. The PIV 5 neuraminidase (NA) domain is monomeric whereas the ectodomain forms a well-defined tetramer. The HN stalk also forms tetramers and higher order oligomers with high alpha-helical content. Together, the data indicate that the globular NA domains form weak intersubunit interactions at the end of the HN stalk tetramer, while stabilizing the stalk and overall oligomeric state of the ectodomain. Electron microscopy of the HN ectodomain reveals flexible arrangements of the NA and stalk domains, which may be important for understanding how these two HN domains impact virus entry.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

Science.gov (United States)

Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

2014-03-01

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

Science.gov (United States)

Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

2014-01-01

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell. PMID:24658080

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter

DEFF Research Database (Denmark)

Norgaard-Nielsen, Kristine; Norregaard, Lene; Hastrup, Hanne

2002-01-01

Increasing evidence suggests that Na(+)/Cl(-)-dependent neurotransmitter transporters exist as homo-oligomeric proteins. However, the functional implication of this oligomerization remains unclear. Here we demonstrate the engineering of a Zn(2+) binding site at the predicted dimeric interface...

Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

Science.gov (United States)

Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

2013-06-24

Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

An engineered allosteric switch in leucine-zipper oligomerization.

Science.gov (United States)

Gonzalez, L; Plecs, J J; Alber, T

1996-06-01

Controversy remains about the role of core side-chain packing in specifying protein structure. To investigate the influence of core packing on the oligomeric structure of a coiled coil, we engineered a GCN4 leucine zipper mutant that switches from two to three strands upon binding the hydrophobic ligands cyclohexane and benzene. In solution these ligands increased the apparent thermal stability and the oligomerization order of the mutant leucine zipper. The crystal structure of the peptide-benzene complex shows a single benzene molecule bound at the engineered site in the core of the trimer. These results indicate that coiled coils are well-suited to function as molecular switches and emphasize that core packing is an important determinant of oligomerization specificity.

Correlation of serum cartilage oligomeric matrix protein (COMP) and interleukin-16 (IL-16) levels with disease severity in primary knee osteoarthritis: A pilot study in a Malaysian population.

Science.gov (United States)

Das Gupta, Esha; Ng, Wei Ren; Wong, Shew Fung; Bhurhanudeen, Abdul Kareem; Yeap, Swan Sim

2017-01-01

The aim of this study was to investigate the correlations between serum cartilage oligomeric matrix protein (COMP), interleukin-16 (IL-16) and different grades of knee osteoarthritis (KOA) in Malaysian subjects. Ninety subjects were recruited comprising 30 with Kellgren-Lawrence (K-L) grade 2 KOA, 27 with K-L grade 3 KOA, 7 with grade 4 KOA, and 30 healthy controls. All subjects completed the Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire. Serum COMP and IL-16 levels were measured using ELISA and their values log transformed to ensure a normal distribution. There was no significant differences in levels of log serum COMP and IL-16 between healthy controls and KOA patients. There were no significant differences in the log serum COMP and IL-16 levels within the different K-L grades in the KOA patients. In KOA patients, log serum IL-16 levels significantly correlated with the WOMAC score (p = 0.001) and its subscales, pain (p = 0.005), stiffness (p = 0.019) and physical function (p<0.0001). Serum IL-16 levels were significantly higher in Malaysian Indians compared to Malays and Chinese (p = 0.024). In this multi-ethnic Malaysian population, there was no difference in serum COMP and IL-16 levels between healthy controls and patients with KOA, nor was there any difference in serum COMP or IL-16 levels across the various K-L grades of KOA. However, there were significant inter-racial differences in serum IL-16 levels.

Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

Science.gov (United States)

Bringolf, Fanny; Herren, Michael; Wyss, Marianne; Vidondo, Beatriz; Langedijk, Johannes P; Zurbriggen, Andreas; Plattet, Philippe

2017-08-15

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity. IMPORTANCE Despite the availability of

Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

Science.gov (United States)

Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

2017-06-21

Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

Ubiquitination of specific mitochondrial matrix proteins

International Nuclear Information System (INIS)

Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

2016-01-01

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Ubiquitination of specific mitochondrial matrix proteins

Energy Technology Data Exchange (ETDEWEB)

Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

2016-06-17

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

DEFF Research Database (Denmark)

Teilum, Kaare; Smith, Melanie H; Schulz, Eike

2009-01-01

remained enigmatic, however, as is the case in other protein-misfolding diseases. Here, we target the critical conformational change that defines the earliest step toward aggregation. Using nuclear spin relaxation dispersion experiments, we identified a short-lived (0.4 ms) and weakly populated (0.......7%) conformation of metal-depleted SOD1 that triggers aberrant oligomerization. This excited state emanates from the folded ground state and is suppressed by metal binding, but is present in both the disulfide-oxidized and disulfide-reduced forms of the protein. Our results pinpoint a perturbed region......Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1). ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have...

Structure and assembly of a paramyxovirus matrix protein.

Science.gov (United States)

Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

2012-08-28

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

Design of a minimal protein oligomerization domain by a structural approach.

Science.gov (United States)

Burkhard, P; Meier, M; Lustig, A

2000-12-01

Because of the simplicity and regularity of the alpha-helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad-repeat peptide stabilized by a complex network of inter- and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly alpha-helical and 100% dimeric tinder physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X-ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native-like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

Science.gov (United States)

Shrivastava, Amulya Nidhi; Redeker, Virginie; Fritz, Nicolas; Pieri, Laura; Almeida, Leandro G; Spolidoro, Maria; Liebmann, Thomas; Bousset, Luc; Renner, Marianne; Léna, Clément; Aperia, Anita; Melki, Ronald; Triller, Antoine

2016-06-01

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263.

Oligomeric forms of the metastasis-related Mts1 (S100A4) protein stimulate neuronal differentiation in cultures of rat hippocampal neurons

DEFF Research Database (Denmark)

Novitskaya, V; Grigorian, M; Kriajevska, M

2000-01-01

protein family. The oligomeric but not the dimeric form of Mts1 strongly induces differentiation of cultured hippocampal neurons. A mutant with a single Y75F amino acid substitution, which stabilizes the dimeric form of Mts1, is unable to promote neurite extension. Disulfide bonds do not play an essential...

Zn(2+) site engineering at the oligomeric interface of the dopamine transporter.

Science.gov (United States)

Norgaard-Nielsen, Kristine; Norregaard, Lene; Hastrup, Hanne; Javitch, Jonathan A; Gether, Ulrik

2002-07-31

Increasing evidence suggests that Na(+)/Cl(-)-dependent neurotransmitter transporters exist as homo-oligomeric proteins. However, the functional implication of this oligomerization remains unclear. Here we demonstrate the engineering of a Zn(2+) binding site at the predicted dimeric interface of the dopamine transporter (DAT) corresponding to the external end of transmembrane segment 6. Upon binding to this site, which involves a histidine inserted in position 310 (V310H) and the endogenous Cys306 within the same DAT molecule, Zn(2+) potently inhibits [(3)H]dopamine uptake. These data provide indirect evidence that conformational changes critical for the translocation process may occur at the interface between two transporter molecules in the oligomeric structure.

Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

Directory of Open Access Journals (Sweden)

Jessica Schmiesing

Full Text Available Glutaric aciduria type 1 (GA1 is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH, which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

Science.gov (United States)

Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

2009-01-01

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

Science.gov (United States)

Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

2016-10-15

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fluorescence reporters for Hfq oligomerization and RNA annealing

Science.gov (United States)

Panja, Subrata; Woodson, Sarah A.

2015-01-01

Fluorescence spectroscopy is a sensitive technique for detecting protein-protein, protein-RNA and RNA-RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer. PMID:25579597

Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

Science.gov (United States)

Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

2017-06-01

The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

Receptor Oligomerization as a Process Modulating Cellular Semiotics

DEFF Research Database (Denmark)

Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

2010-01-01

be another level of quality control that may help maintaining GPCRs rather stable throughout evolution. We propose here receptor oligomerization to be a basic molecular mechanism controlling GPCRs redundancy in many different cell types, and the plasma membrane as the first hierarchical cell structure...... at which selective categorical sensing may occur. Categorical sensing can be seen as the cellular capacity for identifying and ordering complex patterns of mixed signals out of a contextual matrix, i.e., the recognition of meaningful patterns out of ubiquitous signals. In this context, redundancy...

Assembly of the intrinsic factor domains and oligomerization of the protein in the presence of cobalamin

DEFF Research Database (Denmark)

Fedosov, Sergey N; Fedosova, Natalya U; Berglund, Lars

2004-01-01

oligomerized. A mixture of two fragments IF(30) + IF(20) and Cbl produced a firm complex, IF(30+20).Cbl, which could not associate to dimers. In contrast to IF(30+20).Cbl, the saturated full-length monomers IF(50).Cbl dimerized with K(d) approximately 1 microM. We suggest a two-domain organization of the full......-length protein, where two distant units, IF(30) and IF(20), can be assembled only by Cbl. They are connected by a protease-sensitive link, whose native structure is likely to be important for dimerization. However, linkage between two domains is not compulsory for Cbl binding. Advantages of the two...

Detection of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)

Science.gov (United States)

Wang, Jasmine S.-H.; Whitehead, Shawn N.; Yeung, Ken K.-C.

2018-02-01

The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also able to detect the differences in oligomeric composition before and after the incubation/oligomerization step. Such differences in sample composition were also independently confirmed with an in vitro Aβ toxicity study on primary rat cortical neurons. An additional validation was accomplished through removal of oligomers from the sample using molecular weight cutoff filters; the resulting MS data correctly reflected the removal at the expected cutoff points. The results collectively validated the ability of MALDI MS to assess the monomeric/multimeric composition of Aβ samples. [Figure not available: see fulltext.

Electron Microscopy Structural Insights into CPAP Oligomeric Behavior

DEFF Research Database (Denmark)

Alvarez-Cabrera, Ana L; Delgado, Sandra; Gil-Carton, David

2017-01-01

Centrosomal P4.1-associated protein (CPAP) is a cell cycle regulated protein fundamental for centrosome assembly and centriole elongation. In humans, the region between residues 897-1338 of CPAP mediates interactions with other proteins and includes a homodimerization domain. CPAP mutations cause...... into clearly different regular 3D maps (putatively corresponding to dimers and tetramers) and direct observation of individual images representing other complexes of HsCPAP(897-1338) (i.e., putative flexible monomers and higher-order multimers), we report a dynamic oligomeric behavior of this protein, where...... of different oligomers of CPAP, suggesting further insights to understand how this protein works, contributing to the elucidation of control mechanisms for centriole biogenesis....

High Content Analysis of Compositional Heterogeneities to Study GPCR Oligomerization

DEFF Research Database (Denmark)

Walsh, Samuel McEwen

In this thesis I demonstrate how the natural compositional heterogeneities of synthetic and living cell model systems can be used to quantitate the mechanics of G-protein coupled receptor (GPCR) oligomerization with Förster resonance energy transfer (FRET). The thesis is structured around three a...

Characterization of membrane association of Rinderpest virus matrix protein

International Nuclear Information System (INIS)

Subhashri, R.; Shaila, M.S.

2007-01-01

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry

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Amulya Nidhi Shrivastava

2016-06-01

Full Text Available α-Synuclein (α-syn is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD. This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively (doi: 10.15252/embj.201591397 [1]. α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively. For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002263 and doi: 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002263.

Concentration-dependent oligomerization of cross-linked complexes between ferredoxin and ferredoxin–NADP+ reductase

International Nuclear Information System (INIS)

Kimata-Ariga, Yoko; Kubota-Kawai, Hisako; Lee, Young-Ho; Muraki, Norifumi; Ikegami, Takahisa; Kurisu, Genji; Hase, Toshiharu

2013-01-01

Highlights: •Cross-linked complexes of ferredoxin (Fd) and Fd–NADP + reductase form oligomers. •In the crystal structures, Fd- and FNR moieties swap across the molecules. •The complexes exhibit concentration-dependent oligomerization at sub-milimolar order. -- Abstract: Ferredoxin–NADP + reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP + . In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd–FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS–PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd–FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

Science.gov (United States)

Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

2017-06-15

Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still

Escherichia coli PII protein: purification, crystallization and oligomeric structure.

Science.gov (United States)

Vasudevan, S G; Gedye, C; Dixon, N E; Cheah, E; Carr, P D; Suffolk, P M; Jeffrey, P D; Ollis, D L

1994-01-17

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

NARCIS (Netherlands)

Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

1994-01-01

The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

Anesthetic propofol attenuates the isoflurane-induced caspase-3 activation and Aβ oligomerization.

Directory of Open Access Journals (Sweden)

Yiying Zhang

Full Text Available Accumulation and deposition of β-amyloid protein (Aβ are the hallmark features of Alzheimer's disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.

Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

Science.gov (United States)

Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Alejandro Fernández-Velasco, D

2015-08-28

Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.

Conformational detection of p53's oligomeric state by FlAsH Fluorescence

OpenAIRE

Webber, Tawnya M.; Allen, Andrew C.; Ma, Wai Kit; Molloy, Rhett G.; Kettelkamp, Charisse N.; Dow, Caitlin A.; Gage, Matthew J.

2009-01-01

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine ...

Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

Science.gov (United States)

Killian, C. E.; Wilt, F. H.

1996-01-01

In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

OLIGOMERIZATION AND LIQUEFACTION OF ETHYLENE

African Journals Online (AJOL)

oligomerize ethylene gas in a packed bed reactor operated at 100-300°C under apressure of 500psi and ... The gas flow was then switched back to N, gas and temperature controller was simultaneously set to the desired reaction temperature. Once the desired .... be considered non-ideal for ethylene oligomerization.

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

Science.gov (United States)

Imjeti, Naga Salaija; Lebreton, Stéphanie; Paladino, Simona; de la Fuente, Erwin; Gonzalez, Alfonso; Zurzolo, Chiara

2011-12-01

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

Predicting protein-protein interactions from multimodal biological data sources via nonnegative matrix tri-factorization.

Science.gov (United States)

Wang, Hua; Huang, Heng; Ding, Chris; Nie, Feiping

2013-04-01

Protein interactions are central to all the biological processes and structural scaffolds in living organisms, because they orchestrate a number of cellular processes such as metabolic pathways and immunological recognition. Several high-throughput methods, for example, yeast two-hybrid system and mass spectrometry method, can help determine protein interactions, which, however, suffer from high false-positive rates. Moreover, many protein interactions predicted by one method are not supported by another. Therefore, computational methods are necessary and crucial to complete the interactome expeditiously. In this work, we formulate the problem of predicting protein interactions from a new mathematical perspective--sparse matrix completion, and propose a novel nonnegative matrix factorization (NMF)-based matrix completion approach to predict new protein interactions from existing protein interaction networks. Through using manifold regularization, we further develop our method to integrate different biological data sources, such as protein sequences, gene expressions, protein structure information, etc. Extensive experimental results on four species, Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, and Caenorhabditis elegans, have shown that our new methods outperform related state-of-the-art protein interaction prediction methods.

Matrix proteins as centralized organizers of negative-sense RNA virions.

Science.gov (United States)

Liljeroos, Lassi; Butcher, Sarah J

2013-01-01

Matrix proteins are essential components of most negative-sense RNA, enveloped viruses. They serve a wide range of duties ranging from self-driven membrane budding and coordination of other viral components to modulation of viral transcription. The functional similarity between these proteins is striking, despite major differences in their structures. Whereas biochemical and structural studies have partly been hindered by the inherent aggregation properties of these proteins, their cellular functions are beginning to be understood. In this review we summarize the current knowledge on negative-sense RNA virus matrix proteins and their interactions with other viral and cellular proteins. We also discuss the similarities and differences in matrix protein functions between the different families within the negative-sense RNA viruses.

Physiological relevance of plant 2-Cys peroxiredoxin overoxidation level and oligomerization status.

Science.gov (United States)

Cerveau, Delphine; Ouahrani, Djelloul; Marok, Mohamed Amine; Blanchard, Laurence; Rey, Pascal

2016-01-01

Peroxiredoxins are ubiquitous thioredoxin-dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox-dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2-Cys peroxiredoxin (2-Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin-related electron donors, but not on sulfiredoxin, the enzyme reducing the 2-Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2-Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water-stressed Arabidopsis, barley and potato plants, whereas species-dependent up- and down-variations in overoxidation were observed. In contrast, both 2-Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2-Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2-Cys PRX are discussed. © 2015 John Wiley & Sons Ltd.

The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

Science.gov (United States)

Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

2012-08-03

N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

Protein structure estimation from NMR data by matrix completion.

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Li, Zhicheng; Li, Yang; Lei, Qiang; Zhao, Qing

2017-09-01

Knowledge of protein structures is very important to understand their corresponding physical and chemical properties. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods to measure protein structure. In this paper, we propose a two-stage approach to calculate the structure of a protein from a highly incomplete distance matrix, where most data are obtained from NMR. We first randomly "guess" a small part of unobservable distances by utilizing the triangle inequality, which is crucial for the second stage. Then we use matrix completion to calculate the protein structure from the obtained incomplete distance matrix. We apply the accelerated proximal gradient algorithm to solve the corresponding optimization problem. Furthermore, the recovery error of our method is analyzed, and its efficiency is demonstrated by several practical examples.

A Small-Molecule Inhibitor of Bax and Bak Oligomerization Prevents Genotoxic Cell Death and Promotes Neuroprotection.

Science.gov (United States)

Niu, Xin; Brahmbhatt, Hetal; Mergenthaler, Philipp; Zhang, Zhi; Sang, Jing; Daude, Michael; Ehlert, Fabian G R; Diederich, Wibke E; Wong, Eve; Zhu, Weijia; Pogmore, Justin; Nandy, Jyoti P; Satyanarayana, Maragani; Jimmidi, Ravi K; Arya, Prabhat; Leber, Brian; Lin, Jialing; Culmsee, Carsten; Yi, Jing; Andrews, David W

2017-04-20

Aberrant apoptosis can lead to acute or chronic degenerative diseases. Mitochondrial outer membrane permeabilization (MOMP) triggered by the oligomerization of the Bcl-2 family proteins Bax/Bak is an irreversible step leading to execution of apoptosis. Here, we describe the discovery of small-molecule inhibitors of Bax/Bak oligomerization that prevent MOMP. We demonstrate that these molecules disrupt multiple, but not all, interactions between Bax dimer interfaces thereby interfering with the formation of higher-order oligomers in the MOM, but not recruitment of Bax to the MOM. Small-molecule inhibition of Bax/Bak oligomerization allowed cells to evade apoptotic stimuli and rescued neurons from death after excitotoxicity, demonstrating that oligomerization of Bax is essential for MOMP. Our discovery of small-molecule Bax/Bak inhibitors provides novel tools for the investigation of the mechanisms leading to MOMP and will ultimately facilitate development of compounds inhibiting Bax/Bak in acute and chronic degenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

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Yvonne Bartel

2013-01-01

Full Text Available The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21 which appears in about 12% of all de novo acute myeloid leukemias (AMLs. Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2 within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21-positive leukemia.

Stability of human interferon-beta 1: oligomeric human interferon-beta 1 is inactive but is reactivated by monomerization.

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Utsumi, J; Yamazaki, S; Kawaguchi, K; Kimura, S; Shimizu, H

1989-10-05

Human interferon-beta 1 is extremely stable is a low ionic strength solution of pH 2 such as 10 mM HCl at 37 degrees C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-beta 1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per mg protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-beta 1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-beta 1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-beta 1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Characterization of oligomerization of a peptide from the ebola virus glycoprotein by small-angle neutron scattering

International Nuclear Information System (INIS)

Egorov, V. V.; Gorshkov, A. N.; Murugova, T. N.; Vasin, A. V.; Lebedev, D. V.; Isaev-Ivanov, V. V.; Kiselev, O. I.

2016-01-01

Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) studies showed that model peptides QNALVCGLRQ (G33) and QNALVCGLRG (G31) corresponding to region 551–560 of the GP protein of the Sudan Ebola virus are prone to oligomerization in solution. Both peptides can form amyloid-like fibrills. The G33 peptide forms fibrils within one day of incubation, whereas the fibrillogenesis of the G31 peptide is observed only after incubation for several months. The possible role of the observed processes in the pathogenesis and the possibility of applying a combination of the TEM and SANS techniques to search for new compounds that are able to influence the protein oligomerization are discussed

Characterization of oligomerization of a peptide from the ebola virus glycoprotein by small-angle neutron scattering

Energy Technology Data Exchange (ETDEWEB)

Egorov, V. V., E-mail: vlaegur@omrb.pnpi.spb.ru [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Gorshkov, A. N. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation); Murugova, T. N. [Joint Institute for Nuclear Research (Russian Federation); Vasin, A. V. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation); Lebedev, D. V.; Isaev-Ivanov, V. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Kiselev, O. I. [Ministry of Health of the Russian Federation, Research Institute of Influenza (Russian Federation)

2016-01-15

Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) studies showed that model peptides QNALVCGLRQ (G33) and QNALVCGLRG (G31) corresponding to region 551–560 of the GP protein of the Sudan Ebola virus are prone to oligomerization in solution. Both peptides can form amyloid-like fibrills. The G33 peptide forms fibrils within one day of incubation, whereas the fibrillogenesis of the G31 peptide is observed only after incubation for several months. The possible role of the observed processes in the pathogenesis and the possibility of applying a combination of the TEM and SANS techniques to search for new compounds that are able to influence the protein oligomerization are discussed.

Characterization of oligomerization of a peptide from the ebola virus glycoprotein by small-angle neutron scattering

Science.gov (United States)

Egorov, V. V.; Gorshkov, A. N.; Murugova, T. N.; Vasin, A. V.; Lebedev, D. V.; Isaev-Ivanov, V. V.; Kiselev, O. I.

2016-01-01

Transmission electron microscopy (TEM) and small-angle neutron scattering (SANS) studies showed that model peptides QNALVCGLRQ (G33) and QNALVCGLRG (G31) corresponding to region 551-560 of the GP protein of the Sudan Ebola virus are prone to oligomerization in solution. Both peptides can form amyloid-like fibrills. The G33 peptide forms fibrils within one day of incubation, whereas the fibrillogenesis of the G31 peptide is observed only after incubation for several months. The possible role of the observed processes in the pathogenesis and the possibility of applying a combination of the TEM and SANS techniques to search for new compounds that are able to influence the protein oligomerization are discussed.

Binding of triiodothyronine to rat liver nuclear matrix. influence of thyroid hormones on the phosphorylation of nuclear matrix proteins

International Nuclear Information System (INIS)

Adylova, A.T.; Atakhanova, B.A.

1986-01-01

The interaction of thyroid hormones with rat liver nuclear matrix proteins was investigated. It was shown that the nuclear matrix contains sites that bind triiodothyronine with high affinity (K = 1.07.10 9 M -1 ) and limited capacity (the maximum binding capacity is equal to 28 /SUP a/ .5 fmoles of triiodothyronine per 100 ug protein). Electrophoretic identification of the matrix proteins that bind triiodothyronine was performed. The molecular weight of the main triiodothyronine-binding fraction is 50,000-52,000. It was shown that the administration of triiodothyronine to thyroidectomized rats stimulates the phosphorylation of all the protein fractions of the nuclear matrix

Conformational detection of p53's oligomeric state by FlAsH Fluorescence.

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Webber, Tawnya M; Allen, Andrew C; Ma, Wai Kit; Molloy, Rhett G; Kettelkamp, Charisse N; Dow, Caitlin A; Gage, Matthew J

2009-06-19

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein-protein interactions in vivo.

Determination of insoluble avian eggshell matrix proteins

Czech Academy of Sciences Publication Activity Database

Mikšík, Ivan; Sedláková, Pavla; Lacinová, Kateřina; Pataridis, Statis; Eckhardt, Adam

2010-01-01

Roč. 397, č. 1 (2010), s. 205-214 ISSN 1618-2642 R&D Projects: GA MŠk(CZ) 1M0510; GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GA203/08/1428 Institutional research plan: CEZ:AV0Z50110509 Keywords : eggshell proteins * insoluble proteins * matrix proteins Subject RIV: CE - Biochemistry Impact factor: 3.841, year: 2010

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

Science.gov (United States)

Wang, Quan; Serban, Andrew J.; Wachter, Rebekka M.; Moerner, W. E.

2018-03-01

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ˜0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

Endocytic pathways mediating oligomeric Aβ42 neurotoxicity

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Laxton Kevin

2010-05-01

Full Text Available Abstract Background One pathological hallmark of Alzheimer's disease (AD is amyloid plaques, composed primarily of amyloid-β peptide (Aβ. Over-production or diminished clearance of the 42 amino acid form of Aβ (Aβ42 in the brain leads to accumulation of soluble Aβ and plaque formation. Soluble oligomeric Aβ (oAβ has recently emerged to be as a likely proximal cause of AD. Results Here we demonstrate that endocytosis is critical in mediating oAβ42-induced neurotoxicity and intraneuronal accumulation of Aβ. Inhibition of clathrin function either with a pharmacological inhibitor, knock-down of clathrin heavy chain expression, or expression of the dominant-negative mutant of clathrin-assembly protein AP180 did not block oAβ42-induced neurotoxicity or intraneuronal accumulation of Aβ. However, inhibition of dynamin and RhoA by expression of dominant negative mutants reduced neurotoxicity and intraneuronal Aβ accumulation. Pharmacologic inhibition of the dynamin-mediated endocytic pathway by genistein also reduced neurotoxicity. Conclusions These data suggest that dynamin-mediated and RhoA-regulated endocytosis are integral steps for oligomeric Aβ42-induced neurotoxicity and intraneuronal Aβ accumulation.

Molecular basis of coiled-coil oligomerization-state specificity.

Science.gov (United States)

Ciani, Barbara; Bjelic, Saša; Honnappa, Srinivas; Jawhari, Hatim; Jaussi, Rolf; Payapilly, Aishwarya; Jowitt, Thomas; Steinmetz, Michel O; Kammerer, Richard A

2010-11-16

Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains.

Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

Energy Technology Data Exchange (ETDEWEB)

Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

2014-07-01

This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin.

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Bhattacharya, Arpan; Bhowmik, Soumitra; Singh, Amit K; Kodgire, Prashant; Das, Apurba K; Mukherjee, Tushar Kanti

2017-10-10

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Graphene oxide as a protein matrix: influence on protein biophysical properties.

Science.gov (United States)

Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

2015-10-19

This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

RIG-I self-oligomerization is either dispensable or very transient for signal transduction.

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Jade Louber

Full Text Available Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5'ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA, a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.

Reversible peptide oligomerization over nanoscale gold surfaces

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Kazushige Yokoyama

2015-11-01

Full Text Available A selective oligomeric formation of amyloid beta 1-40 (Ab1-40 monomers over a nanogold colloidal surface was investigated. An unfolded Ab1-40 monomer is considered to construct a dimer or trimer based oligomeric form with its hydrophobic segment placing outward under an acidic condition. Under a basic condition, a conformation of Ab is expected to take a folded monomeric form with its hydrophilic segment folded inward, avoiding the networking with residual colloidal particles. The most probable oligomeric form constructed over a 20 nm gold colloidal surface within a 25 ℃ to 65 ℃ temperature range is a dimer based unit and that over 30 or 40 nm gold colloidal surface below 15 ℃ is concluded to be a trimer based unit. However, selective oligomerization was not successfully reproduced under the rest of the conditions. A dipole-induced dipole interaction must cause a flexible structural change between folded and unfolded forms.

Polymorphism and mesomorphism of oligomeric surfactants: effect of the degree of oligomerization.

Science.gov (United States)

Jurašin, D; Pustak, A; Habuš, I; Šmit, I; Filipović-Vinceković, N

2011-12-06

A series of cationic oligomeric surfactants (quaternary dodecyldimethylammonium ions with two, three, or four chains connected by an ethylene spacer at the headgroup level, abbreviated as dimer, trimer, and tetramer) were synthesized and characterized. The influence of the degree of oligomerization on their polymorphic and mesomorphic properties was investigated by means of X-ray diffraction, polarizing optical microscopy, thermogravimetry, and differential scanning calorimetry. All compounds display layered arrangements with interdigitated dodecyl chains. The increase in the degree of oligomerization increases the interlayer distance and decreases the ordering in the solid phase; whereas the dimer sample is fully crystalline with well-developed 3D ordering and the trimer and tetramer crystallize as highly ordered crystal smectic phases. The number of thermal phase transitions and sequence of phases are markedly affected by the number of dodecyl chains. Anhydrous samples exhibit polymorphism and thermotropic mesomorphism of the smectic type, with the exception of the tetramer that displays only transitions at higher temperature associated with decomposition and melting. All hydrated compounds form lyotropic mesophases showing reversible phase transitions upon heating and cooling. The sequence of liquid-crystalline phases for the dimer, typical of concentrated ionic surfactant systems, comprises a hexagonal phase at lower temperatures and a smectic phase at higher temperatures. In contrast, the trimer and tetramer reveal textures of the hexagonal phase. © 2011 American Chemical Society

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

Science.gov (United States)

Fujisawa, Tetsuro; Ueda, Toshifumi; Kameyama, Keiichi; Aso, Yoichi; Ishiguro, Ryo

2013-06-01

The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.

Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

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Itsuki Anzai

2016-08-01

Full Text Available Dominant mutations in Cu/Zn-superoxide dismutase (SOD1 gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS. A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

Interaction between the enamel matrix proteins amelogenin and ameloblastin

International Nuclear Information System (INIS)

Ravindranath, Hanumanth H.; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M.H.

2004-01-01

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [ 3 H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly

Interaction between the enamel matrix proteins amelogenin and ameloblastin.

Science.gov (United States)

Ravindranath, Hanumanth H; Chen, Li-Sha; Zeichner-David, Margaret; Ishima, Rieko; Ravindranath, Rajeswari M H

2004-10-22

Enamel matrix consists of amelogenin and non-amelogenins. Though amelogenin is not involved in nucleation of minerals, the enamel mineralization is impaired when amelogenin or other matrix protein (ameloblastin/enamelin) genes are mutated. We hypothesize that amelogenin may promote enamel mineralization by interacting with the calcium-binding matrix proteins. Specific binding of amelogenin to N-acetylglucosamine (GlcNAc), GlcNAc-mimicking peptides (GMps), and their carrier proteins and the identification of amelogenin-trityrosyl-motif-peptide (ATMP) as a GlcNAc/GMp-binding domain in amelogenin favor the hypothesis. This study tested the interaction of amelogenin with ameloblastin, a carrier of GMp sequence at intermittent sites. Neither GlcNAc nor sialic acids were identified in the recombinant-ameloblastin. Amelogenin bound to recombinant-ameloblastin in both Western blots and in ELISA. More specifically, [(3)H]ATMP bound to both recombinant and native ameloblastins. Dosimetry and Scatchard analyses showed the specific interaction between ATMP and ameloblastin, suggesting that amelogenin may interact with ameloblastin to form a heteromolecular assembly.

Structural adaptation of the subunit interface of oligomeric thermophilic and hyperthermophilic enzymes.

Science.gov (United States)

Maugini, Elisa; Tronelli, Daniele; Bossa, Francesco; Pascarella, Stefano

2009-04-01

Enzymes from thermophilic and, particularly, from hyperthermophilic organisms are surprisingly stable. Understanding of the molecular origin of protein thermostability and thermoactivity attracted the interest of many scientist both for the perspective comprehension of the principles of protein structure and for the possible biotechnological applications through application of protein engineering. Comparative studies at sequence and structure levels were aimed at detecting significant differences of structural parameters related to protein stability between thermophilic and hyperhermophilic structures and their mesophilic homologs. Comparative studies were useful in the identification of a few recurrent themes which the evolution utilized in different combinations in different protein families. These studies were mostly carried out at the monomer level. However, maintenance of a proper quaternary structure is an essential prerequisite for a functional macromolecule. At the environmental temperatures experienced typically by hyper- and thermophiles, the subunit interactions mediated by the interface must be sufficiently stable. Our analysis was therefore aimed at the identification of the molecular strategies adopted by evolution to enhance interface thermostability of oligomeric enzymes. The variation of several structural properties related to protein stability were tested at the subunit interfaces of thermophilic and hyperthermophilic oligomers. The differences of the interface structural features observed between the hyperthermophilic and thermophilic enzymes were compared with the differences of the same properties calculated from pairwise comparisons of oligomeric mesophilic proteins contained in a reference dataset. The significance of the observed differences of structural properties was measured by a t-test. Ion pairs and hydrogen bonds do not vary significantly while hydrophobic contact area increases specially in hyperthermophilic interfaces. Interface

Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

Science.gov (United States)

Turunen, S Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa

2017-11-01

Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

Conformational plasticity of the Ebola virus matrix protein.

Science.gov (United States)

Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

2014-11-01

Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Energy Technology Data Exchange (ETDEWEB)

Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2008-04-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

International Nuclear Information System (INIS)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

Protic Cationic Oligomeric Ionic Liquids of the Urethane Type

DEFF Research Database (Denmark)

Shevchenko, V. V.; Stryutsky, A. V.; Klymenko, N. S.

2014-01-01

Protic oligomeric cationic ionic liquids of the oligo(ether urethane) type are synthesized via the reaction of an isocyanate prepolymer based on oligo(oxy ethylene)glycol with M = 1000 with hexamethylene-diisocyanate followed by blocking of the terminal isocyanate groups with the use of amine...... derivatives of imidazole, pyridine, and 3-methylpyridine and neutralization of heterocycles with ethanesulfonic acid and p-toluenesulfonic acid. The structures and properties of the synthesized oligomeric ionic liquids substantially depend on the structures of the ionic groups. They are amorphous at room...... temperature, but ethanesulfonate imidazolium and pyridinium oligomeric ionic liquids form a low melting crystalline phase. The proton conductivities of the oligomeric ionic liquids are determined by the type of cation in the temperature range 80-120 degrees C under anhydrous conditions and vary within five...

Distance matrix-based approach to protein structure prediction.

Science.gov (United States)

Kloczkowski, Andrzej; Jernigan, Robert L; Wu, Zhijun; Song, Guang; Yang, Lei; Kolinski, Andrzej; Pokarowski, Piotr

2009-03-01

Much structural information is encoded in the internal distances; a distance matrix-based approach can be used to predict protein structure and dynamics, and for structural refinement. Our approach is based on the square distance matrix D = [r(ij)(2)] containing all square distances between residues in proteins. This distance matrix contains more information than the contact matrix C, that has elements of either 0 or 1 depending on whether the distance r (ij) is greater or less than a cutoff value r (cutoff). We have performed spectral decomposition of the distance matrices D = sigma lambda(k)V(k)V(kT), in terms of eigenvalues lambda kappa and the corresponding eigenvectors v kappa and found that it contains at most five nonzero terms. A dominant eigenvector is proportional to r (2)--the square distance of points from the center of mass, with the next three being the principal components of the system of points. By predicting r (2) from the sequence we can approximate a distance matrix of a protein with an expected RMSD value of about 7.3 A, and by combining it with the prediction of the first principal component we can improve this approximation to 4.0 A. We can also explain the role of hydrophobic interactions for the protein structure, because r is highly correlated with the hydrophobic profile of the sequence. Moreover, r is highly correlated with several sequence profiles which are useful in protein structure prediction, such as contact number, the residue-wise contact order (RWCO) or mean square fluctuations (i.e. crystallographic temperature factors). We have also shown that the next three components are related to spatial directionality of the secondary structure elements, and they may be also predicted from the sequence, improving overall structure prediction. We have also shown that the large number of available HIV-1 protease structures provides a remarkable sampling of conformations, which can be viewed as direct structural information about the

Different domains are critical for oligomerization compatibility of different connexins

Science.gov (United States)

MARTÍNEZ, Agustín D.; MARIPILLÁN, Jaime; ACUÑA, Rodrigo; MINOGUE, Peter J.; BERTHOUD, Viviana M.; BEYER, Eric C.

2011-01-01

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies. PMID:21348854

Domain organizations of modular extracellular matrix proteins and their evolution.

Science.gov (United States)

Engel, J

1996-11-01

Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

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Jones Candace A

2011-10-01

Full Text Available Abstract Background Striatin, a putative protein phosphatase 2A (PP2A B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM, which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3 protein, the mammalian Mps one binder (MOB homolog, Mob3/phocein, the mammalian sterile 20-like (Mst kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via

Mixed-matrix membrane adsorbers for protein separation

NARCIS (Netherlands)

Avramescu, M.E.; Borneman, Z.; Wessling, M.

2003-01-01

The separation of two similarly sized proteins, bovine serum albumin (BSA) and bovine hemoglobin (Hb) was carried out using a new type of ion-exchange mixed-matrix adsorber membranes. The adsorber membranes were prepared by incorporation of various types of Lewatit ion-exchange resins into an

In vivo extracellular matrix protein expression by human periodontal ...

African Journals Online (AJOL)

It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

Thermo-mechanical characterization of a monochlorophenyl, hepta isobutyl polyhedral oligomeric silsesquioxane/polystyrene composite

International Nuclear Information System (INIS)

Blanco, Ignazio; Bottino, Francesco A.; Cicala, Gianluca; Cozzo, Giulia; Latteri, Alberta; Recca, Antonino

2014-01-01

The thermal and mechanical properties of a monochlorophenyl, hepta isobutyl Polyhedral Oligomeric Silsesquioxane/Polystyrene (ph,hib-POSS/PS) composite were studied and compared with those of pristine polymer. ph,hib-POSS/PS system was prepared by solubilization and precipitation of Polystyrene (PS) in the presence of POSS. Scanning Electron Microscopy (SEM) was performed to check the distribution of the filler in the polymer matrix. Dynamic Mechanical Analysis (DMA) was carried out to measure viscoelastic properties of solid samples. Degradations were carried out into a thermobalance and the obtained thermogravimetric (TG) and differential thermogravimetric (DTG) curves were discussed and interpreted

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

Science.gov (United States)

Ito, Mikako; Ohno, Kinji

2018-02-20

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Science.gov (United States)

Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

2016-04-22

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Oligomerization in health and disease

CERN Document Server

Giraldo, Jesus

2013-01-01

This special volume of Progress in Molecular Biology and Translational Science focuses on oligomerization in health and disease. Contributions from leading authorities Informs and updates on all the latest developments in the field.

Supplementary Material for: A new mode of SAM domain mediated oligomerization observed in the CASKIN2 neuronal scaffolding protein

KAUST Repository

Smirnova, Ekaterina; Kwan, Jamie; Siu, Ryan; Gao, Xin; Zoidl, Georg; Demeler, Borries; Saridakis, Vivian; Donaldson, Logan

2016-01-01

Abstract Background CASKIN2 is a homolog of CASKIN1, a scaffolding protein that participates in a signaling network with CASK (calcium/calmodulin-dependent serine kinase). Despite a high level of homology between CASKIN2 and CASKIN1, CASKIN2 cannot bind CASK due to the absence of a CASK Interaction Domain and consequently, may have evolved undiscovered structural and functional distinctions. Results We demonstrate that the crystal structure of the Sterile Alpha Motif (SAM) domain tandem (SAM1-SAM2) oligomer from CASKIN2 is different than CASKIN1, with the minimal repeating unit being a dimer, rather than a monomer. Analytical ultracentrifugation sedimentation velocity methods revealed differences in monomer/dimer equilibria across a range of concentrations and ionic strengths for the wild type CASKIN2 SAM tandem and a structure-directed double mutant that could not oligomerize. Further distinguishing CASKIN2 from CASKIN1, EGFP-tagged SAM tandem proteins expressed in Neuro2a cells produced punctae that were distinct both in shape and size. Conclusions This study illustrates a new way in which neuronal SAM domains can assemble into large macromolecular assemblies that might concentrate and amplify synaptic responses.

Purification, crystallization and preliminary crystallographic analysis of Est-Y29: a novel oligomeric β-lactamase

International Nuclear Information System (INIS)

Kim, SeungBum; Joo, Sangbum; Yoon, Sangyoung; Kim, Sungsoo; Moon, Jongkook; Ryu, Yeonwoo; Kim, Kyeong Kyu; Kim, T. Doohun

2009-01-01

Est-Y29, a novel oligomeric class C β-lactamase from a metagenomic library, was crystallized in space group I4 1 and diffraction data were collected to 1.49 Å resolution. β-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The β-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C β-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. The recombinant protein was expressed in Escherichia coli with an N-terminal 6×His tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49 Å resolution using synchrotron radiation

Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis

International Nuclear Information System (INIS)

Rogolinski, J.; Widlak, P.; Rzeszowska-Wolny, J.

1996-01-01

Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rats testis cells. Starting from 2 weeks the young to adult animal showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some enriched in spermatocytes and spermatids and obtained after fractionation of cells of adult animal by the velocity sedimentation technique. (author). 21 refs, 5 figs

Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.

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Yi Zhang

Full Text Available Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.

Oligomerization interface of RAGE receptor revealed by MS-monitored hydrogen deuterium exchange.

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Ewa Sitkiewicz

Full Text Available Activation of the receptor for advanced glycation end products (RAGE leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.

In vivo extracellular matrix protein expression by human periodontal ...

African Journals Online (AJOL)

ONOS

2010-08-23

Aug 23, 2010 ... Extracellular matrix proteins (ECM) are described as molecular regulators of these events. ..... zation and adhesive interaction of cells (Yamada, 1983). .... periodontal ligament fibroblasts after simulation of orthodontic force.

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

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Stephen C Graham

2008-12-01

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

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

Science.gov (United States)

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

A single peroxisomal targeting signal mediates matrix protein import in diatoms.

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Nicola H Gonzalez

Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

The L-L oligomerization domain resides at the very N-terminus of the sendai virus L RNA polymerase protein

International Nuclear Information System (INIS)

Cevik, Bayram; Smallwood, Sherin; Moyer, Sue A.

2003-01-01

The Sendai virus RNA-dependent RNA polymerase is composed of the L and P proteins. We previously showed that the L protein gives intragenic complementation and forms an oligomer where the L-L interaction site mapped to the N-terminal half of the protein (S. Smallwood et al., 2002, Virology, 00, 000-000). We now show that L oligomerization does not depend on P protein and progressively smaller N-terminal fragments of L from amino acids (aa) 1-1146 through aa 1-174 all bind wild-type L. C-terminal truncations up to aa 424, which bind L, can complement the transcription defect in an L mutant altered at aa 379, although these L truncation mutants do not bind P. The fragment of L comprising aa 1-895, furthermore, acts as a dominant-negative mutant to inhibit transcription of wild-type L. N-terminal deletions of aa 1-189 and aa 1-734 have lost the ability to form the L-L complex as well as the L-P complex, although they still bind C protein. These data are consistent with the L-L interaction site residing in aa 1-174. Site-directed mutations in the N-terminal 347 aa, of L which abolish P binding, do not affect L-L complex formation, so while the L and P binding sites on L are overlapping they are mediated by different amino acids. The N-terminal portions of L with aa 1-424, aa 1-381, and to a lesser extent aa 1-174, can complement the transcription defect in an L mutant altered at aa 77-81, showing their L-L interaction is functional

Biophysical characterization of GPCR oligomerization

DEFF Research Database (Denmark)

Mathiasen, Signe

level, and revealed the existence of several dimerization interfaces, each with specific kinetics. Finally we investigated how a property of the membrane solubilizing GPCRs affected oligomerization. We observed a dramatic decrease in oligomer stability with increasing geometrical membrane curvature. We...

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

Science.gov (United States)

Tayou, Junior; Wang, Qiang; Jang, Geeng-Fu; Pronin, Alexey N; Orlandi, Cesare; Martemyanov, Kirill A; Crabb, John W; Slepak, Vladlen Z

2016-04-22

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of early changes of cartilage biomarkers following ...

African Journals Online (AJOL)

Hamdy Khamis Koryem

2014-08-15

Aug 15, 2014 ... resulting in structural, biochemical and mechanical changes that can progress from pre-clinical, to pre-radiographic .... blasts/chondrocytes) that produce an extracellular matrix of ... to represent an adequate index of the rate of type II collagen ... collagenous proteins (e.g. cartilage oligomeric matrix protein,.

Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

Science.gov (United States)

Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

2016-01-01

Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

Oligomeric protein complexes of apolipoproteins stabilize the internal fluid environment of organism in redfins of the Tribolodon genus [Pisces; Cypriniformes, Cyprinidae].

Science.gov (United States)

Andreeva, Alla M; Serebryakova, Marina V; Lamash, Nina E

2017-06-01

One of the most important functions of plasma proteins in vertebrates is their participation in osmotic homeostasis in the organism. Modern concepts about plasma proteins and their capillary filtration are based on a model of large monomeric proteins that are able to penetrate the interstitial space. At the same time, it was revealed that a considerable amount of oligomeric complexes are present in the low-molecular-weight (LM) protein fraction in the extracellular fluids of fishes. The functions of these complexes are unknown. In the present study, we investigated the LM-fraction proteins in the plasma and interstitial fluid (IF) of redfins of the genus Tribolodon. This fish alternatively spends parts of its life cycle in saline and fresh waters. We identified the protein Wap65, serpins and apolipoproteins in this fraction. By combining the methods of 2D-E under native and denaturing conditions with MALDI, we demonstrated that only apolipoproteins formed complexes. We showed that serum apolipoproteins (АроА-I, Аро-14) were present in the form of homooligomeric complexes that were dissociated with the release of monomeric forms of proteins in the course of capillary filtration to IF. Dissociation of homooligomers is not directly correlated with the change in salinity but is correlated with seasonal dynamics. We found that there was a significant decrease in the total protein concentration in IF relative to plasma. Therefore, we suggested that dissociation of homooligomeric complexes from various apolipoproteins supports the isoosmoticity of extracellular fluids relative to capillary wall stabilization through a fluid medium in fish. Copyright © 2017 Elsevier Inc. All rights reserved.

A major protein component of the Bacillus subtilis biofilm matrix.

Science.gov (United States)

Branda, Steven S; Chu, Frances; Kearns, Daniel B; Losick, Richard; Kolter, Roberto

2006-02-01

Microbes construct structurally complex multicellular communities (biofilms) through production of an extracellular matrix. Here we present evidence from scanning electron microscopy showing that a wild strain of the Gram positive bacterium Bacillus subtilis builds such a matrix. Genetic, biochemical and cytological evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multicellular communities. Extracellular complementation experiments revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.

octamethyl-polyhedral oligomeric silsesquioxanes nanocomposites ...

Indian Academy of Sciences (India)

In this study, biodegradable poly(p-dioxanone) (PPDO)/octamethyl-polyhedral oligomeric silsesquioxanes (ome-POSS) nanocomposites were fabricated by the simple solution casting method with various ome-POSS loadings. Scanning electron microscopic observations indicate that ome-POSS is well dispersed in the ...

Suppression of matrix protein synthesis in endothelial cells by herpes simplex virus is not dependent on viral protein synthesis

International Nuclear Information System (INIS)

Kefalides, N.A.

1986-01-01

The synthesis of matrix proteins by human endothelial cells (EC) in vitro was studied before and at various times after infection with Herpes Simplex virus Type 1 (HSV-1) or 2 (HSV-2). Monolayers of EC were either mock-infected or infected with virus for 1 hr at a multiplicity infection (MOI) of 5 to 20 at 37 0 C. Control and infected cultures were pulse-labeled for 1 or 2 hrs with either [ 14 C]proline or [ 35 S]methionine. Synthesis of labeled matrix proteins was determined by SDS-gel electrophoresis. Suppression of synthesis of fibronectin, Type IV collagen and thrombospondin began as early as 2 hrs and became almost complete by 10 hrs post-infection. The degree of suppression varied with the protein and the virus dose. Suppression of Type IV collagen occurred first followed by that of fibronectin and then thrombospondin. Infection of EC with UV irradiated HSV-1 or HSV-2 resulted in suppression of host-cell protein synthesis as well as viral protein synthesis. Infection with intact virus in the presence of actinomycin-D resulted in suppression of both host-cell and viral protein synthesis. The data indicate that infection of EC with HSV leads to suppression of matrix protein synthesis which does not depend on viral protein synthesis

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation*

OpenAIRE

Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H.; Krulwich, Terry A.; Ito, Masahiro

2010-01-01

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA–MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na+(Li+)/H+ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resista...

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Science.gov (United States)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

LRPPRC is a mitochondrial matrix protein that is conserved in metazoans

International Nuclear Information System (INIS)

Sterky, Fredrik H.; Ruzzenente, Benedetta; Gustafsson, Claes M.; Samuelsson, Tore; Larsson, Nils-Goeran

2010-01-01

Research highlights: → LRPPRC orthologs are restricted to metazoans. → LRPPRC is imported to the mitochondrial matrix. → No evidence of nuclear isoform. -- Abstract: LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Efficient light-emitting devices based on platinum-complexes-anchored polyhedral oligomeric silsesquioxane materials

KAUST Repository

Yang, Xiaohui

2010-08-24

The synthesis, photophysical, and electrochemical characterization of macromolecules, consisting of an emissive platinum complex and carbazole moieties covalently attached to a polyhedral oligomeric silsesquioxane (POSS) core, is reported. Organic light-emitting devices based on these POSS materials exhibit a peak external quantum efficiency of ca. 8%, which is significantly higher than that of the analogous devices with a physical blend of the platinum complexes and a polymer matrix, and they represent noticeable improvement in the device efficiency of solution-processable phosphorescent excimer devices. Furthermore, the ratio of monomer and excimer/aggregate electroluminescent emission intensity, as well as the device efficiency, increases as the platinum complex moiety presence on the POSS macromolecules decreases. © 2010 American Chemical Society.

Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

Science.gov (United States)

Getzenberg, R H; Coffey, D S

1990-09-01

The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

Science.gov (United States)

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.

Directory of Open Access Journals (Sweden)

Jie Gao

Full Text Available The optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE, little is known about the characteristics of optineurin oligomers. Here, we aimed to analyze the oligomeric state of optineurin and factors affecting oligomerization, such as environmental stimuli or mutations in OPTN. Using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, we demonstrated that both endogenous and transfected optineurin exist as oligomers, rather than monomers, in NIH3T3 cells. We also applied an in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and found that the optineurin oligomers were localized diffusely in the cytoplasm. Optineurin oligomers were usually detected as a single band of a size equal to that of the optineurin monomer upon SDS-PAGE, while an additional protein band of a larger size was observed when cells were treated with H2O2. We showed that larger protein complex is optineurin oligomers by immunoprecipitation and termed it covalent optineurin oligomers. In cells expressing OPTN bearing the most common glaucoma-associated mutation, E50K, covalent oligomers were formed even without H2O2 stimulation. Antioxidants inhibited the formation of E50K-induced covalent oligomers to various degrees. A series of truncated constructs of OPTN was used to reveal that covalent oligomers may be optineurin trimers and that the ubiquitin-binding domain is essential for formation of these trimers. Our results indicated that optineurin trimers may be the basic unit of these oligomers. The oligomeric state can be affected by many factors that induce covalent bonds, such as H2O2 or E50K, as demonstrated here; this provides novel insights into the pathogenicity of E50K. Furthermore

Collagen and related extracellular matrix proteins in atherosclerotic plaque development.

Science.gov (United States)

Shami, Annelie; Gonçalves, Isabel; Hultgårdh-Nilsson, Anna

2014-10-01

The structure, composition and turnover of the extracellular matrix (ECM) as well as cell-matrix interactions are crucial in the developing atherosclerotic plaque. There is a need for further insight into specific proteins in the ECM and their functions in the developing plaque, and during the last few years a number of publications have highlighted this very important field of research. These novel findings will be addressed in the present review. This review covers literature focused on collagen and ECM proteins interacting with collagen, and what their roles may be in plaque development. Acute myocardial infarction and stroke are common diseases that cause disability and mortality, and the underlying mechanism is often the rupture of a vulnerable atherosclerotic plaque. The vascular ECM and the tissue repair in the atherosclerotic lesion are important players in plaque progression. Understanding how specific proteins in the ECM interact with cells in the plaque and affect the fate of the plaque can lead to new treatments for cardiovascular disease.

Transparent cellulose/polyhedral oligomeric silsesquioxane nanocomposites with enhanced UV-shielding properties.

Science.gov (United States)

Feng, Ye; Zhang, Jinming; He, Jiasong; Zhang, Jun

2016-08-20

The solubility of eight types of polyhedral oligomeric silsesquioxane (POSS) derivatives in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl) and the dispersion of POSS in cellulose matrix were examined. Only a special POSS containing both aminophenyl and nitrophenyl groups (POSS-AN, NH2:NO2=2:6) was selected to prepare nanocomposites, because of its good solubility in AmimCl and high stability during the preparation process. POSS-AN nanoparticles were uniformly dispersed in a cellulose matrix with a size of 30-40nm, and so the resultant cellulose/POSS-AN nanocomposite films were transparent. The mechanical properties of the films achieved a maximum tensile strength of 190MPa after addition of 2wt% POSS-AN. Interestingly, all of the cellulose/POSS-AN films exhibited high UV-absorbing capability. For the 15wt% cellulose/POSS-AN film, the transmittance of UVA (315-400nm) and UVB (280-315nm) was only 9.1% and nearly 0, respectively. The UV aging and shielding experiments showed that the transparent cellulose/POSS-AN nanocomposite films possessed anti-UV aging and UV shielding properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

DEFF Research Database (Denmark)

Gannagé, Monique; Dormann, Dorothee; Albrecht, Randy

2009-01-01

Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we...... demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient...... for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell...

An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity.

Science.gov (United States)

Pacheco, Sabino; Gómez, Isabel; Sánchez, Jorge; García-Gómez, Blanca-Ines; Soberón, Mario; Bravo, Alejandra

2017-10-15

Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity. Copyright © 2017 American Society for Microbiology.

Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

Science.gov (United States)

Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

1994-11-25

The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

Directory of Open Access Journals (Sweden)

Srsen Vlastimil

2009-04-01

Full Text Available Abstract Background Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. Results We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. Conclusion Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

Assessment of Escherichia coli selenophosphate synthetase oligomeric states by analytical ultracentrifugation and small angle X-ray scattering

Energy Technology Data Exchange (ETDEWEB)

Silva, I.R.; Faim, F.M.; Oliveira Neto, M.; Thiemann, O.H. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil); Borges, J.C. [Universidade de Sao Paulo (IQSC/USP), Sao Carlos, SP (Brazil). Inst. de Quimica

2012-07-01

Full text: Selenium is an essential micronutrient for many organisms and is present in selenium-containing proteins as selenocysteine (Sec) and RNAs as selenouridine. Specific selenium incorporation into selenoproteins and RNAs requires the generation of a biologically active selenium donor compound, selenophosphate, which is produced from the activation of selenide with adenosine 5-triphosphate (ATP) in a reaction catalyzed by Selenophosphate Synthetase (SELD). Therefore, SELD is a key enzyme of the selenium pathway in the cell. The Escherichia coli SELD open reading frame was cloned into pET28a (Novagen) expression vector and the recombinant protein was over expressed in Escherichia coli BL21(DE3) strain. In order to purify the protein, we used metal-chelate affinity chromatography followed by a gel filtration step. Analytical Ultracentrifugation (AUC) and Small Angle X-ray Scattering (SAXS) were employed to study the oligomeric states of the soluble protein. The results of AUC revealed dimer-tetramer and tetramer-octamer equilibrium at low concentrations of protein, with dissociation constants of 70 2 and 560 40 M, respectively. Moreover, the SAXS results pointed the oligomeric state of the protein at higher concentrations as predominantly dimeric and the p(r) and the SAXS envelope revealed the SELD as elongated. We also performed initial crystallization trials with protein samples at 7 mg/ml in 96-well sitting-drop crystallization plates at room temperature using a crystallization robot. Needle crystals appeared after some days. X-ray diffraction for these crystals were tested in the MX2 beamline at the Brazilian Synchrotron Laboratory (LNLS Campinas). We are now working to improve these crystals in order to obtain suitable crystals for structure determination. (author)

An Overview of the Medical Applications of Marine Skeletal Matrix Proteins

Directory of Open Access Journals (Sweden)

M. Azizur Rahman

2016-09-01

Full Text Available In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP. Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.

Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

Science.gov (United States)

Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

2011-03-01

We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

Synthesis and characterization of polyhedral oligomeric titanized silsesquioxane: A new biocompatible cage like molecule for biomedical application

Energy Technology Data Exchange (ETDEWEB)

Yahyaei, Hossein [Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran (Iran, Islamic Republic of); Mohseni, Mohsen, E-mail: mmohseni@aut.ac.ir [Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, P.O. Box 15875-4413, Tehran (Iran, Islamic Republic of); Ghanbari, Hossein [Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran (Iran, Islamic Republic of); Messori, Massimo [Dipartimento di Ingegneria ‘Enzo Ferrari’, Università di Modena e Reggio Emilia, Modena (Italy)

2016-04-01

Organic–inorganic hybrid materials have shown improved properties to be used as biocompatible coating in biomedical applications. Polyhedral oligomeric silsesquioxane (POSS) containing coatings are among hybrid materials showing promising properties for these applications. In this work an open cage POSS has been reacted with a titanium alkoxide to end cap the POSS molecule with titanium atom to obtain a so called polyhedral oligomeric metalized silsesquioxane (POMS). The synthesized POMS was characterized by FTIR, RAMAN and UV–visible spectroscopy as well as {sup 29}Si NMR and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) techniques. Appearance of peaks at 920 cm{sup −1} in FTIR and 491 cm{sup −1} and 1083 cm{sup −1} in Raman spectra confirmed Si–O–Ti linkage formation. It was also demonstrated that POMS was in a monomeric form. To evaluate the biocompatibility of hybrids films, pristine POSS and synthesized POMS were used in synthesis of a polycarbonate urethane polymer. Results revealed that POMS containing hybrid, not only had notable thermal and mechanical stability compared to POSS containing one, as demonstrated by DSC and DMTA analysis, they also showed controlled surface properties in such a manner that hydrophobicity and biocompatibility were both reachable to give rise to improved cell viability in presence of human umbilical vein endothelial cells (HUVEC) and MRC-5 cells. - Highlight: • Polyhedral Oligomeric Metalized Silsesquioxane (POMS) based on titanium was synthesized. • POMS can improve mechanical properties of polyurethane. • POMS increases hydrophobicity of polyurethane. • POMS is a unique nanocage to enhance biocompatibility of polyurethane.

Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

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Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2010-10-01

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

Topical application of amelogenin extracellular matrix protein in non-healing venous ulcers

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Burçin Abud

2014-12-01

Full Text Available Background and Design: Treatment of chronic venous ulcers of the lower extremity is still an important difficulty. The principal treatment of these ulcers includes compression therapy, local wound care and surgery. Unresponsiveness to these standard treatments is a frequent situation with negative effects on life quality and reductions in personal productivity. Therefore, there is a need for new applications to increase the effectiveness of treatment in treatment-resistant cases. In the present study, we retrospectively evaluated the results of topical application of amelogenin extracellular matrix protein in resistant venous ulcers. Materials and Methods: We analyzed the records of patients with treatment-resistant venous ulceration who were treated with amelogenin extracellular matrix protein between June 2011 and December 2012.. Results: 26 patients (21 male and 5 female with a total number of 28 ulcers (24 patients with 1 ulcer, 2 patients with two ulcers were evaluated. The patients were treated with topically applied amelogenin extracellular matrix protein and regional four bandage compression. Bandages were changed weekly. Each cure continued for six weeks. In fourteen patients (15 ulcers, we observed a complete healing by the end of the first cure. In another twelve cases (13 ulcers, the same period resulted with a reduction in wound diameter. We continued to the second cure for these patients. By the end of the second cure, complete healing was achieved in five cases (6 ulcers. Conclusion: Topical application of amelogenin extracellular matrix protein may be considered as an effective therapeutic choice for refractory venous ulcers.

Molecular events in matrix protein metabolism in the aging kidney

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Sataranatarajan, Kavithalakshmi; Feliers, Denis; Mariappan, Meenalakshmi M.; Lee, Hak Joo; Lee, Myung Ja; Day, Robert T.; Yalamanchili, Hima Bindu; Choudhury, Goutam G.; Barnes, Jeffrey L.; Van Remmen, Holly; Richardson, Arlan; Kasinath, Balakuntalam S.

2018-01-01

Summary We explored molecular events associated with aging-induced matrix changes in the kidney. C57BL6 mice were studied in youth, middle age, and old age. Albuminuria and serum cystatin C level (an index of glomerular filtration) increased with aging. Renal hypertrophy was evident in middle-aged and old mice and was associated with glomerulomegaly and increase in mesangial fraction occupied by extracellular matrix. Content of collagen types I and III and fibronectin was increased with aging; increment in their mRNA varied with the phase of aging. The content of ZEB1 and ZEB2, collagen type I transcription inhibitors, and their binding to the collagen type Iα2 promoter by ChIP assay also showed age-phase-specific changes. Lack of increase in mRNA and data from polysome assay suggested decreased degradation as a potential mechanism for kidney collagen type I accumulation in the middle-aged mice. These changes occurred with increment in TGFβ mRNA and protein and activation of its SMAD3 pathway; SMAD3 binding to the collagen type Iα2 promoter was also increased. TGFβ-regulated microRNAs (miRs) exhibited selective regulation. The renal cortical content of miR-21 and miR-200c, but not miR-192, miR-200a, or miR-200b, was increased with aging. Increased miR-21 and miR-200c contents were associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively. These data show that aging is associated with complex molecular events in the kidney that are already evident in the middle age and progress to old age. Agephase-specific regulation of matrix protein synthesis occurs and involves matrix protein-specific transcriptional and post-transcriptional mechanisms. PMID:23020145

Structure-properties relationships of polyhedral oligomeric silsesquioxane (POSS filled PS nanocomposites

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J. J. Schwab

2012-07-01

Full Text Available The polyhedral oligomeric silsesquioxane (POSS additivated polystyrene (PS based nanocomposites were prepared by melt processing and the structure-properties relationships of the POSS-PS systems were compared to those of the neat PS. In order to investigate the effect of these structural parameters on the final properties of the polymer nanocomposites, five different kinds of POSS samples were used, in particular, POSS with different inorganic cage and with different organic pendent groups. The rheological investigation suggests clearly that the POSS acts as a plasticizer and that the processability of the PS was positively modified. The affinity between the POSS samples and the PS matrix was estimated by the calculated theoretical solubility parameters, considering the Hoy’s method and by morphology analysis. Minor difference between the solubility parameter of POSS and the matrix means better compatibility and no aggregation tendency. Furthermore, the POSS loading leads to a decrease of the rigidity, of the glass transition temperature and of the damping factor of the nanocomposite systems. The loading of different POSS molecules with open cage leads to a more pronounced effect on all the investigated properties that the loading of the POSS molecules with closed cage. Moreover, the melt properties are significantly influenced by the type of inorganic framework, by the type of the pendent organic groups and by the interaction between the POSS organic groups and the host matrix, while, the solid state properties appears to be influenced more by the kind of cage.

The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

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Chang, Chung-ke [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Wu, Tzong-Huah [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Biochemistry, Academia Sinica, Taipei 115, Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan (China); Wu, Chu-Ya [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Graduate Institute of Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan (China); Chiang, Ming-hui; Toh, Elsie Khai-Woon [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Hsu, Yin-Chih; Lin, Ku-Feng [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China); Liao, Yu-heng [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Huang, Tai-huang, E-mail: bmthh@gate.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan (China); Department of Physics, National Taiwan Normal University, Taipei 106, Taiwan (China); Huang, Joseph Jen-Tse, E-mail: jthuang@chem.sinica.edu.tw [Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan (China)

2012-08-24

Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

Dielectric properties of polyhedral oligomeric silsesquioxane (POSS)-based nanocomposites at 77k

International Nuclear Information System (INIS)

Pan, Ming-Jen; Gorzkowski, Edward; McAllister, Kelly

2011-01-01

The goal of this study is to develop dielectric nanocomposites for high energy density applications at liquid nitrogen temperature by utilizing a unique nano-material polyhedral oligomeric silsesquioxanes (POSS). A POSS molecule is consisted of a silica cage core with 8 silicon and 12 oxygen atoms and organic functional groups attached to the corners of the cage. In this study, we utilize POSS for the fabrication of nanocomposites both as a silica nanoparticle filler to enhance the breakdown strength and as a surfactant for effective dispersion of high permittivity ceramic nanoparticles in a polymer matrix. The matrix materials selected for the study are polyvinylidene fluoride (PVDF) and poly(methyl methacrylate) (PMMA). The ceramic nanoparticles are barium strontium titanate (BST 50/50) and strontium titanate. The dielectric properties of the solution-cast nanocomposites films were correlated to the composition and processing conditions. We determined that the addition of POSS did not provide enhanced dielectric performance in PVDF- and PMMA-based materials at either room temperature or 77K. In addition, we found that the dielectric breakdown strength of PMMA is lower at 77K than at room temperature, contradicting literature data.

Photo-polymerization of photocurable resins containing polyhedral oligomeric silsesquioxane methacrylate

International Nuclear Information System (INIS)

Lin, Ho-May; Wu, Shi-Yin; Chang, Feng-Chih; Yen, Ying-Chieh

2011-01-01

Photocurable resins, bisphenol A propoxylate glycerolate diacrylate (BPA-PGDA, containing two hydroxyl) and bisphenol A propoxylate diacrylate (BPA-PDA), with fixed photoinitiator (Irgacure 907) concentration and various contents of methacrylisobutyl polyhedral oligomeric silsesquioxane (MI-POSS) were prepared and characterized by FTIR spectroscopy, scanning electron microscope and differential photocalorimetry. The MI-POSS molecules form crystals or aggregated particles in the cured resin matrix. The BPA-PGDA series photocurable resins show higher viscosity and lower photo-polymerization reactivity than the BPA-PDA series resins. The photo-polymerization rate and conversion of BPA-PGDA series are improved with increasing MI-POSS content. On the contrary, the photo-polymerization behavior of BPA-PDA series photocurable resins remains nearly unchanged by the addition of MI-POSS. Hydrogen-bonding interaction between the hydroxyl of BPA-PGDA and the siloxane of MI-POSS tends to attract and concentrate these acrylate double bonds around MI-POSS particles and thus enhances the photo-polymerization rate and conversion.

The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

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Davey Megan J

2010-09-01

Full Text Available Abstract Background Minichromosome maintenance proteins (Mcm 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7. Results Here, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer. Conclusions The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

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Brast, Sabine; Grabner, Alexander; Sucic, Sonja; Sitte, Harald H; Hermann, Edwin; Pavenstädt, Hermann; Schlatter, Eberhard; Ciarimboli, Giuliano

2012-03-01

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

A S52P mutation in the 'α-crystallin domain' of Mycobacterium leprae HSP18 reduces its oligomeric size and chaperone function.

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Nandi, Sandip K; Rehna, Elengikal A A; Panda, Alok K; Shiburaj, Sugathan; Dharmalingam, Kuppamuthu; Biswas, Ashis

2013-12-01

Mycobacterium leprae HSP18 is a small heat shock protein (sHSP). It is a major immunodominant antigen of M. leprae pathogen. Previously, we have reported the existence of two M. leprae HSP18 variants in various leprotic patients. One of the variants has serine at position 52, whereas the other one has proline at the same position. We have also reported that HSP18 having proline at position 52 (HSP18P(52)) is a nonameric protein and exhibits chaperone function. However, the structural and functional characterization of wild-type HSP18 having serine at position 52 (HSP18S(52)) is yet to be explored. Furthermore, the implications of the S52P mutation on the structure and chaperone function of HSP18 are not well understood. Therefore, we cloned and purified these two HSP18 variants. We found that HSP18S(52) is also a molecular chaperone and an oligomeric protein. Intrinsic tryptophan fluorescence and far-UV CD measurements revealed that the S52P mutation altered the tertiary and secondary structure of HSP18. This point mutation also reduced the oligomeric assembly and decreased the surface hydrophobicity of HSP18, as revealed by HPLC and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid binding studies, respectively. Mutant protein was less stable against thermal and chemical denaturation and was more susceptible towards tryptic cleavage than wild-type HSP18. HSP18P(52) had lower chaperone function and was less effective in protecting thermal killing of Escherichia coli than HSP18S(52). Taken together, our data suggest that serine 52 is important for the larger oligomerization and chaperone function of HSP18. Because both variants differ in stability and function, they may have different roles in the survival of M. leprae in infected hosts. © 2013 FEBS.

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

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Fu, Yuan; Lin, Hongyu; Wisitpitthaya, Somsinee; Blessing, William A; Aye, Yimon

2014-11-24

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

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Case, Heather B; Dickenson, Nicholas E

2018-04-17

Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta

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Moreno, Herman; Yu, Eunah; Pigino, Gustavo; Hernandez, Alejandro I.; Kim, Natalia; Moreira, Jorge E.; Sugimori, Mutsuyuki; Llinás, Rodolfo R.

2009-01-01

Early Alzheimer's disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)Aβ42, but not oAβ40 or extracellular oAβ42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oAβ42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD. PMID:19304802

Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells.

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Narula, Shifa; Tandon, Simran; Singh, Shrawan Kumar; Tandon, Chanderdeep

2016-11-01

Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. The renal epithelial cells (MDCK) were exposed to 200μg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

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Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level

A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP.

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Serrano, Arnaldo L; Lomont, Justin P; Tu, Ling-Hsien; Raleigh, Daniel P; Zanni, Martin T

2017-11-22

Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F 23 G 24 A 25 I 26 L 27 region of hIAPP, which starts from a random coil structure, evolves into ordered β-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 μM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.

Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins

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Shibata, Toshio; Maki, Kouki; Hadano, Jinki; Fujikawa, Takumi; Kitazaki, Kazuki; Koshiba, Takumi; Kawabata, Shun-ichiro

2015-01-01

Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes. PMID:26506243

Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins.

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Toshio Shibata

2015-10-01

Full Text Available Transglutaminase (TG catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.

Discovery of Nanomolar Desmuramylpeptide Agonists of the Innate Immune Receptor Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Possessing Immunostimulatory Properties.

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Gobec, Martina; Tomašič, Tihomir; Štimac, Adela; Frkanec, Ruža; Trontelj, Jurij; Anderluh, Marko; Mlinarič-Raščan, Irena; Jakopin, Žiga

2018-04-12

Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.

Oligomeric adiponectin forms and their complexes in the blood of healthy donors and patients with type 2 diabetes mellitus.

Science.gov (United States)

Kogan, Alexander E; Filatov, Vladimir L; Kolosova, Olga V; Katrukha, Ivan A; Mironova, Ekaterina V; Zhuravleva, Natalya S; Nagibin, Oleg A; Kara, Andrei N; Bereznikova, Anastasiya V; Katrukha, Alexey G

2013-01-01

Adiponectin (Adn) is a protein that circulates in the blood in several oligomeric forms, namely low-, medium-, and high-molecular-weight forms. Adn may serve as a risk factor for type 2 diabetes mellitus (T2DM). The aims of this work were (1) to produce monoclonal antibodies (MAbs) specific to different Adn oligomeric forms, (2) to design immunoassays suitable for measuring the Adn forms present in human blood, and (3) to investigate the changes in Adn forms that occur in patients with T2DM. Gel filtration, fluoroimmunoassays, and Western blotting were utilized as major techniques in this study. MAbs recognizing various oligomeric forms of Adn were obtained. Complexes between Adn and complement component C1q and between the low molecular weight form of Adn and albumin were described in human blood. A decrease in the total Adn and Adn-albumin complex levels in the blood of patients with T2DM and no difference in the levels of the Adn-C1q complex in comparison with healthy volunteers were demonstrated. An Adn94-Adn63 fluoroimmunoassay was selected as the technique that most accurately measured the mass ratio of Adn oligomers in blood samples, and an Adn214-Adn27 assay that measured the low-molecular-weight form of Adn only.

Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

International Nuclear Information System (INIS)

Montrose, Kristopher; Krissansen, Geoffrey W.

2014-01-01

Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC

Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus

Energy Technology Data Exchange (ETDEWEB)

Montrose, Kristopher; Krissansen, Geoffrey W., E-mail: gw.krissansen@auckland.ac.nz

2014-10-31

Highlights: • A novel proteolysis targeting chimeric molecule (PROTAC) to treat hepatitis B. • The PROTAC antagonizes and destroys the X-protein of the hepatitis B virus. • The PROTAC is a fusion of the X-protein oligomerization and instability domains. • The oligomerization domain is a dominant-negative inhibitor of X-protein function. • X-protein-targeting PROTACs have potential to prevent hepatocellular carcinoma. - Abstract: The X-protein of the hepatitis B virus (HBV) is essential for virus infection and contributes to the development of HBV-induced hepatocellular carcinoma (HCC), a disease which causes more than one million deaths each year. Here we describe the design of a novel PROTAC (proteolysis targeting chimeric molecule) capable of simultaneously inducing the degradation of the X-protein, and antagonizing its function. The PROTAC was constructed by fusing the N-terminal oligomerization and C-terminal instability domains of the X-protein to each other, and rendering them cell-permeable by the inclusion of a polyarginine cell-penetrating peptide (CPP). It was predicted that the oligomerization domain would bind the X-protein, and that the instability domain would cause the X-protein to be targeted for proteasomal degradation. Addition of the PROTAC to HepG2 liver cancer cells, engineered to express full-length and C-terminally truncated forms of the X-protein, resulted in the degradation of both forms of the X-protein. A cell-permeable stand-alone form of the oligomerization domain was taken up by HepG2 cells, and acted as a dominant-negative inhibitor, causing inhibition of X-protein-induced apoptosis. In summary, the PROTAC described here induces the degradation of the X-protein, and antagonizes its function, and warrants investigation in a preclinical study for its ability to prevent or treat HBV infection and/or the development of HCC.

Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83.

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Anne Müller

Full Text Available The murine G-protein coupled receptor 83 (mGPR83 is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR such as the ghrelin receptor (GHSR or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83 by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation.

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

Science.gov (United States)

Kasimsetty, Sashi G; Shigeoka, Alana A; Scheinok, Andrew A; Gavin, Amanda L; Ulevitch, Richard J; McKay, Dianne B

2017-08-01

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2 -/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2 -/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2 -/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation. Copyright © 2017 by The American Association of Immunologists, Inc.

One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins

Czech Academy of Sciences Publication Activity Database

Doležal, Michal; Zábranský, Aleš; Hrabal, R.; Ruml, T.; Pichová, Iva; Rumlová, Michaela

2013-01-01

Roč. 92, č. 1 (2013), s. 94-99 ISSN 1046-5928 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : matrix protein * mouse mammary tumor virus * murine leukemia virus * myristoylation * N-myristoyltransferase * retrovirus Subject RIV: CE - Biochemistry Impact factor: 1.508, year: 2013

Thermal Degradation Mechanism of a Thermostable Polyester Stabilized with an Open-Cage Oligomeric Silsesquioxane

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Yolanda Bautista

2017-12-01

Full Text Available A polyester composite was prepared through the polymerization of an unsaturated ester resin with styrene and an open-cage oligomeric silsesquioxane with methacrylate groups. The effect of the open-cage oligomeric silsesquioxane on the thermal stability of the thermostable polyester was studied using both thermogravimetric analysis and differential thermal analysis. The results showed that the methacryl oligomeric silsesquioxane improved the thermal stability of the polyester. The decomposition mechanism of the polyester/oligomer silsesquioxane composite was proposed by Fourier transform infrared spectroscopy (FTIR analysis of the volatiles.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

Science.gov (United States)

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Oligomerization process

Science.gov (United States)

Smith, L.A. Jr.; Hearn, D.; Jones, E.M. Jr.

1991-03-26

A liquid phase process is described for oligomerization of C[sub 4] and C[sub 5] isoolefins or the etherification thereof with C[sub 1] to C[sub 6] alcohols wherein the reactants are contacted in a reactor with a fixed bed acid cation exchange resin catalyst at an LHSV of 5 to 20, pressure of 0 to 400 psig and temperature of 120 to 300 F wherein the improvement is the operation of the reactor at a pressure to maintain the reaction mixture at its boiling point whereby at least a portion but less than all of the reaction mixture is vaporized. By operating at the boiling point and allowing a portion of the reaction mixture to vaporize, the exothermic heat of reaction is dissipated by the formation of more boil up and the temperature in the reactor is controlled. 2 figures.

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

Science.gov (United States)

Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

2013-01-01

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

Science.gov (United States)

Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

2014-01-01

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

Vitamin K Intake and Plasma Desphospho-Uncarboxylated Matrix Gla-Protein Levels in Kidney Transplant Recipients

NARCIS (Netherlands)

Boxma, P.Y.; Berg, van den E.; Geleijnse, J.M.; Laverman, G.D.; Schurgers, L.J.; Vermeer, C.; Kema, I.P.; Muskiet, F.A.J.; Navis, G.; Bakker, S.J.L.; Borst, de M.H.

2012-01-01

Vitamin K is essential for activation of ¿-carboxyglutamate (Gla)-proteins including the vascular calcification inhibitor matrix Gla-protein (MGP). Insufficient vitamin K intake leads to production of uncarboxylated, mostly inactive proteins and contributes to an increased cardiovascular risk. In

Calcinosis in juvenile dermatomyositis : a possible role for the vitamin K-dependent protein matrix Gla protein

NARCIS (Netherlands)

Van Summeren, M. J. H.; Spliet, W. G. M.; Van Royen-Kerkhof, A.; Vermeer, C.; Lilien, M.; Kuis, W.; Schurgers, L. J.

Objectives. The aims of the present study were to investigate whether the calcification inhibitor matrix Gla protein (MGP) is expressed in muscle biopsies of patients with juvenile dermatomyositis (JDM), and whether different forms of MGP are differentially expressed in JDM patients with and without

Reactive bay functionalized perylene monoimide-polyhedral oligomeric silsesquioxane organic electronic dye

Directory of Open Access Journals (Sweden)

Wangatia Lodrick Makokha

2015-03-01

Full Text Available Aggregation-induced quenching is particularly detrimental in perylene diimides, which are characterized by a near-unity fluorescence quantum yield in solution but are far less emissive in the solid state. Previously, perylene diimide has been improved by linking it to the inorganic cage of polyhedral oligomeric silsesquioxanes. As a further study on perylene diimidepolyhedral oligomeric silsesquioxanes, we report on a double functionalized molecular structure, which can be used for substitution at the bay area and as a side group in other materials. Typical solution absorption and emission features of the perylene diimide fragment have been observed in this new reactive perylene diimide-polyhedral oligomeric silsesquioxane. Moreover, reduced stacking during aggregation and spherical particles exhibiting solid fluorescence have been obtained. Organic semiconducting material with enhanced solid state photophysical properties, like solid fluorescence is a subject of great interest owing to its possible high-tech applications in optoelectronic devices.

Biocatalytically Oligomerized Epicatechin with Potent and Specific Anti-proliferative Activity for Human Breast Cancer Cells

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Ramaswamy Nagarajan

2008-11-01

Full Text Available Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP as a catalyst yielding water-soluble oligo(epicatechins. The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents.

Directory of Open Access Journals (Sweden)

Anil S Thakur

2007-10-01

Full Text Available Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed.We tested the effect of nine potential heterogenous nucleating agents on crystallization of ten test proteins in a sparse matrix screen. Several nucleating agents induced crystal formation under conditions where no crystallization occurred in the absence of the nucleating agent. Four nucleating agents: dried seaweed; horse hair; cellulose and hydroxyapatite, had a considerable overall positive effect on crystallization success. This effect was further enhanced when these nucleating agents were used in combination with each other.Our results suggest that the addition of heterogeneous nucleating agents increases the chances of crystal formation when using sparse matrix screens.

Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

DEFF Research Database (Denmark)

Houen, G.; Olsen, D.T.; Hansen, P.R.

2003-01-01

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

Protective Effects of Testosterone on Presynaptic Terminals against Oligomeric β-Amyloid Peptide in Primary Culture of Hippocampal Neurons

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Chi-Fai Lau

2014-01-01

Full Text Available Increasing lines of evidence support that testosterone may have neuroprotective effects. While observational studies reported an association between higher bioavailable testosterone or brain testosterone levels and reduced risk of Alzheimer’s disease (AD, there is limited understanding of the underlying neuroprotective mechanisms. Previous studies demonstrated that testosterone could alleviate neurotoxicity induced by β-amyloid (Aβ, but these findings mainly focused on neuronal apoptosis. Since synaptic dysfunction and degeneration are early events during the pathogenesis of AD, we aim to investigate the effects of testosterone on oligomeric Aβ-induced synaptic changes. Our data suggested that exposure of primary cultured hippocampal neurons to oligomeric Aβ could reduce the length of neurites and decrease the expression of presynaptic proteins including synaptophysin, synaptotagmin, and synapsin-1. Aβ also disrupted synaptic vesicle recycling and protein folding machinery. Testosterone preserved the integrity of neurites and the expression of presynaptic proteins. It also attenuated Aβ-induced impairment of synaptic exocytosis. By using letrozole as an aromatase antagonist, we further demonstrated that the effects of testosterone on exocytosis were unlikely to be mediated through the estrogen receptor pathway. Furthermore, we showed that testosterone could attenuate Aβ-induced reduction of HSP70, which suggests a novel mechanism that links testosterone and its protective function on Aβ-induced synaptic damage. Taken together, our data provide further evidence on the beneficial effects of testosterone, which may be useful for future drug development for AD.

Caught in self-interaction: evolutionary and functional mechanisms of protein homooligomerization

International Nuclear Information System (INIS)

Hashimoto, Kosuke; Nishi, Hafumi; Bryant, Stephen; Panchenko, Anna R

2011-01-01

Many soluble and membrane proteins form homooligomeric complexes in a cell which are responsible for the diversity and specificity of many pathways, may mediate and regulate gene expression, activity of enzymes, ion channels, receptors, and cell adhesion processes. The evolutionary and physical mechanisms of oligomerization are very diverse and its general principles have not yet been formulated. Homooligomeric states may be conserved within certain protein subfamilies and might be important in providing specificity to certain substrates while minimizing interactions with other unwanted partners. Moreover, recent studies have led to a greater awareness that transitions between different oligomeric states may regulate protein activity and provide the switch between different pathways. In this paper we summarize the biological importance of homooligomeric assemblies, physico-chemical properties of their interfaces, experimental and computational methods for their identification and prediction. We particularly focus on homooligomer evolution and describe the mechanisms to develop new specificities through the formation of different homooligomeric complexes. Finally, we discuss the possible role of oligomeric transitions in the regulation of protein activity and compile a set of experimental examples with such regulatory mechanisms

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

Science.gov (United States)

Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

2012-12-01

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

Methyl methacrylate oligomerically-modified clay and its poly(methyl methacrylate) nanocomposites

International Nuclear Information System (INIS)

Zheng Xiaoxia; Jiang, David D.; Wilkie, Charles A.

2005-01-01

A methyl methacrylate oligomerically-modified clay was used to prepare poly(methyl methacrylate) clay nanocomposites by melt blending and the effect of the clay loading level on the modified clay and corresponding nanocomposite was studied. These nanocomposites were characterized by X-ray diffraction, transmission electron microscopy, thermogravimetric analysis and cone calorimetry. The results show a mixed intercalated/delaminated morphology with good nanodispersion. The compatibility between the methylacrylate-subsituted clay and poly(methyl methacrylate) (PMMA) are greatly improved compared to other oligomerically-modified clays

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

International Nuclear Information System (INIS)

Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

1987-01-01

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

International Nuclear Information System (INIS)

Vasile, Eliza; Oka, Toshihiko; Ericsson, Maria; Nakamura, Nobuhiro; Krieger, Monty

2006-01-01

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

International Nuclear Information System (INIS)

Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

2011-01-01

With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

Catalyst-controlled oligomerization for the collective synthesis of polypyrroloindoline natural products.

Science.gov (United States)

Jamison, Christopher R; Badillo, Joseph J; Lipshultz, Jeffrey M; Comito, Robert J; MacMillan, David W C

2017-12-01

In nature, many organisms generate large families of natural product metabolites that have related molecular structures as a means to increase functional diversity and gain an evolutionary advantage against competing systems within the same environment. One pathway commonly employed by living systems to generate these large classes of structurally related families is oligomerization, wherein a series of enzymatically catalysed reactions is employed to generate secondary metabolites by iteratively appending monomers to a growing serial oligomer chain. The polypyrroloindolines are an interesting class of oligomeric natural products that consist of multiple cyclotryptamine subunits. Herein we describe an iterative application of asymmetric copper catalysis towards the synthesis of six distinct oligomeric polypyrroloindoline natural products: hodgkinsine, hodgkinsine B, idiospermuline, quadrigemine H and isopsychotridine B and C. Given the customizable nature of the small-molecule catalysts employed, we demonstrate that this strategy is further amenable to the construction of quadrigemine H-type alkaloids not isolated previously from natural sources.

Substitution of proline32 by α-methylproline preorganizes β2-microglobulin for oligomerization but not for aggregation into amyloids.

Science.gov (United States)

Torbeev, Vladimir; Ebert, Marc-Olivier; Dolenc, Jozica; Hilvert, Donald

2015-02-25

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human β2-microglobulin (β2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a β2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]β2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type β2m, [MePro32]β2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type β2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]β2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial β2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of β2m amyloid structures.

Dimerization of a flocculent protein from Moringa oleifera: experimental evidence and in silico interpretation.

Science.gov (United States)

Pavankumar, Asalapuram R; Kayathri, Rajarathinam; Murugan, Natarajan A; Zhang, Qiong; Srivastava, Vaibhav; Okoli, Chuka; Bulone, Vincent; Rajarao, Gunaratna K; Ågren, Hans

2014-01-01

Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein-protein docking studies and binding free energy calculations. The structure of MO2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.

Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

Science.gov (United States)

Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

2016-02-23

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Fingolimod phosphate attenuates oligomeric amyloid β-induced neurotoxicity via increased brain-derived neurotrophic factor expression in neurons.

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Yukiko Doi

Full Text Available The neurodegenerative processes that underlie Alzheimer's disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P receptor (S1PR agonist fingolimod phosphate (FTY720-P-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer's disease.

Structural symmetry and protein function.

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Goodsell, D S; Olson, A J

2000-01-01

The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of

Controlling amyloid-beta peptide(1-42) oligomerization and toxicity by fluorinated nanoparticles.

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Saraiva, Ana M; Cardoso, Isabel; Pereira, M Carmo; Coelho, Manuel A N; Saraiva, Maria João; Möhwald, Helmuth; Brezesinski, Gerald

2010-09-03

The amyloid-beta peptide (Abeta) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Abeta42. We show here that fluorinated and hydrogenated NPs with different abilities to change Abeta42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS-PAGE. Fluorinated NPs, which promote an increase in alpha-helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Abeta42 into an alpha-helical-enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.

Glucosamine but not ibuprofen alters cartilage turnover in osteoarthritis patients in response to physical training

DEFF Research Database (Denmark)

Petersen, Susanne Germann; Saxne, T; Heinegard, D

2010-01-01

OBJECTIVE: To investigate changes in levels of serum cartilage oligomeric matrix protein (COMP) and urine c-telopeptide of type-2 collagen (CTX-II) as markers for cartilage turnover in patients with osteoarthritis (OA) of the knee, in response to muscle strength training in combination with treat......OBJECTIVE: To investigate changes in levels of serum cartilage oligomeric matrix protein (COMP) and urine c-telopeptide of type-2 collagen (CTX-II) as markers for cartilage turnover in patients with osteoarthritis (OA) of the knee, in response to muscle strength training in combination......). RESULTS: All three groups increased their muscle strength following 12 weeks of strength training (Preduced in the glucosamine-treated group after the training period (P=0.012), whereas they did not change in the two other groups. Glucosamine reduced COMP statistically...

Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

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Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

2014-01-01

Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

Structure and expression of an unusually acidic matrix protein of pearl oyster shells

International Nuclear Information System (INIS)

Tsukamoto, Daiki; Sarashina, Isao; Endo, Kazuyoshi

2004-01-01

We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp) 2-10 sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata

Peeping into human renal calcium oxalate stone matrix: characterization of novel proteins involved in the intricate mechanism of urolithiasis.

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Kanu Priya Aggarwal

Full Text Available BACKGROUND: The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal-membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin-Darby Canine Kidney (MDCK renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated. RESULTS: Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSIONS: We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone

Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry

DEFF Research Database (Denmark)

Andersen, Jens S.; Svensson, B; Roepstorff, P

1996-01-01

Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy is presen......Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy...... is presented encompassing protein characterization prior to and after cloning of the corresponding gene....

Effect Of Oligomeric Enteral Nutrition On Symptoms Of Acute Radiation Enteritis

International Nuclear Information System (INIS)

Dubinsky, P.

2008-01-01

Radiotherapy of abdominal and pelvic tumours is frequently associated with acute radiation enteritis. Predominant symptoms include diarrhea, watery stools, abdominal pain, nausea and vomiting. There are very few effective interventions available for this condition. Enteral oligomeric nutrition has been used in bowel diseases with functional failure similar to radiation enteritis. The aim of presented work was to observe occurrence of symptoms of radiation enteritis in patients undergoing abdominal or pelvic radiotherapy. Apart from diet and pharmacological therapy, oral oligomeric enteral nutrition (Peptisorb Powder Nutricia) at the dose of 1000 - 2000 ml per day was administered for minimum of 4 days. Planned period of administration was 14 days and longer. Symptoms of radiation enteritis were evaluated at the beginning and in the end of administration. Prevalence of all evaluated symptoms of radiation enteritis was decreased and difference was statistically significant for diarrhea, watery stools, abdominal pain, nausea and vomiting. The use of evaluated oligomeric nutritional support might, in conjunction with pharmacotherapy and diet, alleviate symptoms of acute radiation enteritis and maintain nutritional status of patients. (author)

Growth of thin films of low molecular weight proteins by matrix assisted pulsed laser evaporation (MAPLE)

DEFF Research Database (Denmark)

Matei, Andreea; Schou, Jørgen; Constantinescu, C.

2011-01-01

Thin films of lysozyme and myoglobin grown by matrix assisted pulsed laser evaporation (MAPLE) from a water ice matrix have been investigated. The deposition rate of these two low molecular weight proteins (lysozyme: 14307 amu and myoglobin: 17083 amu) exhibits a maximum of about 1–2 ng/cm2 per....... The results for lysozyme demonstrate that the fragmentation rate of the proteins during the MAPLE process is not influenced by the pH of the water solution prior to freezing....

Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men.

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Churchward-Venne, Tyler A; Snijders, Tim; Linkens, Armand M A; Hamer, Henrike M; van Kranenburg, Janneau; van Loon, Luc J C

2015-07-01

The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

Science.gov (United States)

Inforzato, Antonio; Rivieccio, Vincenzo; Morreale, Antonio P; Bastone, Antonio; Salustri, Antonietta; Scarchilli, Laura; Verdoliva, Antonio; Vincenti, Silvia; Gallo, Grazia; Chiapparino, Caterina; Pacello, Lucrezia; Nucera, Eleonora; Serlupi-Crescenzi, Ottaviano; Day, Anthony J; Bottazzi, Barbara; Mantovani, Alberto; De Santis, Rita; Salvatori, Giovanni

2008-04-11

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

Identification and characterization of stable membrane protein complexes

NARCIS (Netherlands)

Spelbrink, R.E.J.

2007-01-01

Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

Thermodynamics of protein folding: a random matrix formulation.

Science.gov (United States)

Shukla, Pragya

2010-10-20

The process of protein folding from an unfolded state to a biologically active, folded conformation is governed by many parameters, e.g. the sequence of amino acids, intermolecular interactions, the solvent, temperature and chaperon molecules. Our study, based on random matrix modeling of the interactions, shows, however, that the evolution of the statistical measures, e.g. Gibbs free energy, heat capacity, and entropy, is single parametric. The information can explain the selection of specific folding pathways from an infinite number of possible ways as well as other folding characteristics observed in computer simulation studies. © 2010 IOP Publishing Ltd

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

Science.gov (United States)

Liu, Xiaojun; Zeng, Shimei; Dong, Shaojian; Jin, Can; Li, Jiale

2015-01-01

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.

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.

Membrane and inclusion body targeting of lyssavirus matrix proteins.

Science.gov (United States)

Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

2013-02-01

Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

DC-SIGN neck domain is a pH-sensor controlling oligomerization: SAXS and hydrodynamic studies of extracellular domain.

Science.gov (United States)

Tabarani, Georges; Thépaut, Michel; Stroebel, David; Ebel, Christine; Vivès, Corinne; Vachette, Patrice; Durand, Dominique; Fieschi, Franck

2009-08-07

DC-SIGN is a C-type lectin receptor of dendritic cells and is involved in the early stages of numerous infectious diseases. DC-SIGN is organized into a tetramer enabling multivalent interaction with pathogens. Once formed, the DC-SIGN-pathogen complex can be internalized into compartments of increasing acidity. We have studied the pH dependence of the oligomerization state and conformation of the entire extracellular domain and neck region. We present evidence for equilibrium between the monomeric and tetrameric states of the extracellular domain, which exhibits a marked dependence with respect to both pH and ionic strength. Using solution x-ray scattering we have obtained a molecular envelope of the extracellular domain in which a model has been built. Our results highlight the central role of the neck domain in the pH-sensitive control of the oligomerization state, in the extended conformation of the protein, and in carbohydrate recognition domain organization and presentation. This work opens new insight into the molecular mechanism of ligand release and points to new avenues to block the first step of this important infection pathway.

Matrix-assisted laser-desorption-ionization mass spectrometry of proteins using a free-electron laser

International Nuclear Information System (INIS)

Cramer, R.; Hillenkamp, F.; Haglund, R.

1995-01-01

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is one of the most promising techniques for spectral fingerprinting large molecules, such as proteins, oligonucleotides and carbohydrates. In the usual implementation of this technique, the analyte molecule is dissolved in an aromatic liquid matrix material which resonantly absorbs ultraviolet laser light. Resonant absorption by π-π* transitions volatilizes the matrix and initiates subsequent charge transfer to the analyte molecules, which are detected by time-of-flight mass spectrometry. Recent MALDI-MS studies with Er:YAG (2.94 μm) and CO 2 4 (9.4-10.6 μm) lasers suggest that them is significant unexplored potential for mass spectrometry of macromolecules, including oligonucleotide, in the mid-infrared. Preliminary experiments show that it is possible to capitalize on the rich rovibronic absorption spectrum of virtually all organics to initiate resonant desorption in matrix material over the entire range of pH values. However, the mechanism of charge transfer is particularly problematic for infrared MALDI because of the low photon energy. In this paper, we report the results of MALI-MS studies on small proteins using the Vanderbilt FEL and several matrix materials. Proteins with masses up to roughly 6,000 amu were detected with high resolution in a linear time-of-flight mass spectrometer. By varying the pulse duration using a broadband Pockels cell, we have been able to compare the results of relatively long (5 μs) and short (0.1 μs) irradiation on the desorption and ionization processes. Compared to uv-MALDI spectra of identical analytes obtained with a nitrogen laser (337 nm) in the same time-of-flight spectrometer, the infrared results appear to show that the desorption and ionization process goes on over a somewhat longer time scale

Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

DEFF Research Database (Denmark)

Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

2013-01-01

The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...... extracellular matrix (ECM) proteins known as neoepitopes, which are then released into the circulation....

S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins.

Directory of Open Access Journals (Sweden)

Michael R Dorwart

Full Text Available While the bacterial mechanosensitive channel of large conductance (MscL is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL is a pentamer, while the S. aureus protein (SaMscL forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8E(5 detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.

S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins.

Science.gov (United States)

Dorwart, Michael R; Wray, Robin; Brautigam, Chad A; Jiang, Youxing; Blount, Paul

2010-12-07

While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8)E(5) detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.

Postsynaptic density protein 95 in the striosome and matrix compartments of the human neostriatum.

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Ryoma eMorigaki

2015-11-01

Full Text Available The human neostriatum consists of two functional subdivisions referred to as the striosome (patch and matrix compartments. The striosome-matrix dopamine systems play a central role in cortico-thalamo-basal ganglia circuits, and their involvement is thought to underlie the genesis of multiple movement and behavioral disorders, and of drug addiction. Human neuropathology also has shown that striosomes and matrix have differential vulnerability patterns in several striatal neurodegenerative diseases. Postsynaptic density protein 95 (PSD-95, also known as DLG4, is a major scaffolding protein in the postsynaptic densities of dendritic spines. PSD-95 is now known to negatively regulate not only N-methyl-D-aspartate glutamate signaling, but also dopamine D1 signals at sites of postsynaptic transmission. Accordingly, a neuroprotective role for PSD-95 against dopamine D1 receptor (D1R-mediated neurotoxicity in striatal neurodegeneration also has been suggested. Here, we used a highly sensitive immunohistochemistry technique to show that in the human neostriatum, PSD-95 is differentially concentrated in the striosome and matrix compartments, with a higher density of PSD-95 labeling in the matrix compartment than in the striosomes. This compartment-specific distribution of PSD-95 was strikingly complementary to that of D1R. In addition to the possible involvement of PSD-95-mediated synaptic function in compartment-specific dopamine signals, we suggest that the striosomes might be more susceptible to D1R-mediated neurotoxicity than the matrix compartment. This notion may provide new insight into the compartment-specific vulnerability of MSNs in striatal neurodegeneration.

Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

Science.gov (United States)

Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

Science.gov (United States)

Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

2015-10-01

Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

Cobalt Oxide on N-Doped Carbon for 1-Butene Oligomerization to Produce Linear Octenes

Energy Technology Data Exchange (ETDEWEB)

Zhao, Dongting [Department; Xu, Zhuoran [Department; Chada, Joseph P. [Department; Carrero, Carlos A. [Department; Rosenfeld, Devon C. [The Dow Chemical Company, 2301 N. Brazosport Boulevard, Freeport, Texas 77541-3257, United States; Rogers, Jessica L. [The Dow Chemical Company, 2301 N. Brazosport Boulevard, Freeport, Texas 77541-3257, United States; Hermans, Ive [Department; Huber, George W. [Department

2017-10-02

Cobalt oxide supported on N-doped carbon catalysts were investigated for 1-butene oligomerization. The materials were synthesized by treating activated carbon with nitric acid and subsequently with NH3 at 200, 400, 600, and 800 °C, followed by impregnation with cobalt. The 1-butene oligomerization selectivity increased with ammonia treatment temperature of the carbon support. The oligomerization selectivity of cobalt oxide on N-doped carbon synthesized at 800 °C (800A-CoOx/N-C) is 2.6 times higher than previously reported cobalt oxide on N-doped carbon synthesized with NH4OH (2A-CoOx/N-C). Over 70% of the butene dimers were linear C8 olefins for all catalysts. The oligomerization selectivity increased with 1-butene conversion. The catalysts were characterized by elemental analysis, N2 adsorption, X-ray diffraction (XRD), X-ray absorption spectroscopy (XAS), and X-ray photoelectron spectroscopy (XPS). The nitrogen content of the catalysts increases with ammonia treatment temperature as confirmed by elemental analysis. The surface content of pyridinic nitrogen with a binding energy of 398.4 ± 0.1 eV increased with ammonia treatment temperature as evidenced by deconvolution of N 1s XPS spectra.

Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

Science.gov (United States)

Yan, Shao-Min; Wu, Guang

2009-12-01

The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry for the Investigation of Proteins and Peptides

Science.gov (United States)

Burnum, Kristin E.; Frappier, Sara L.; Caprioli, Richard M.

2008-07-01

Mass spectrometry (MS) is an excellent technology for molecular imaging because of its high data dimensionality. MS can monitor thousands of individual molecular data channels measured as mass-to-charge (m/z). We describe the use of matrix-assisted laser desorption/ionization (MALDI) MS for the image analysis of proteins, peptides, lipids, drugs, and metabolites in tissues. We discuss the basic instrumentation and sample preparation methods needed to produce high-resolution images and high image reproducibility. Matrix-addition protocols are briefly discussed along with normal operating procedures, and selected biological and medical applications of MALDI imaging MS are described. We give examples of both two- and three-dimensional imaging, including normal mouse embryo implantation, sperm maturation in mouse epididymis, protein distributions in brain sections, protein alterations as a result of drug administration, and protein changes in brain due to neurodegeneration and tumor formation. Advantages of this technology and future challenges for its improvement are discussed.

Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

Energy Technology Data Exchange (ETDEWEB)

Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.; Neuman, B.W.; Buchmeier, M.J.; Stevens, R.C.; Kuhn, P.; /Scripps Res. Inst.

2007-07-12

Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17A (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Human α4β2 nicotinic acetylcholine receptor as a novel target of oligomeric α-synuclein.

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Qiang Liu

Full Text Available Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5'-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM analyses, we find that only large (>4 nm oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

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C.L. Vituri

2000-08-01

Full Text Available The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM. Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5% and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase and laminin (4.8-fold increase when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Beyond the Protein Matrix : Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

NARCIS (Netherlands)

Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

2013-01-01

A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP(+) and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like

Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

Science.gov (United States)

Qiao, F; Moss, A; Kupfer, G M

2001-06-29

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.

Catalytic oligomerization of terminal alkynes promoted by organo-f-complexes

International Nuclear Information System (INIS)

Straub, T.; Haskel, A.; Eisen, M.S.

1995-01-01

Organoactinides of the type Cp* 2 AcMe 2 (Cp*=C 5 Me 5 ; Ac=Th, U) are active catalyst precursors for the oligomerization of terminal alkynes HC triple-bond CR (R=alkyl, aryl, SiMe 3 ). The regioselectivity and the extent of oligomerization strongly depend on the alkyne substituent R, whereas the catalytic reactivity is similar for 1 and 2. In the presence of one of these organoactinides, for example, HCCSiMe 3 regioselectively oligomerizes to the head-to-tail dimer 3 (5%) and the trimer 4 (95%). 1 and 2 react with the terminal alkynes, releasing methane, to the corresponding bisacetylide complexes which are active species and in the catalytic reactions. The bisacetylide complex (η 5 -C 5 Me 5 ) 2 U(CCPh) 2 was identified by proton NMR spectroscopy. Subsequent insertion of alkyne molecules in the actinide-carbon σ-bonds leads to the formation of actinide-alkenyl complexes. The turnover limiting step is the release of the organic oligomer from the actinide-organyl complex. A species of the latter has been spectroscopically characterized in the trimerization reaction of HCCSiMe 3 . In this poster, the catalytic reactivity of the actinide alkyls 1 and 2 with various mono-substituted alkynes as well as the spectroscopic characterization of the key organometallic intermediate complexes in the catalytic cycle and a detailed mechanistic discussion are given

Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

Science.gov (United States)

Echtay, Karim S; Murphy, Michael P; Smith, Robin A J; Talbot, Darren A; Brand, Martin D

2002-12-06

Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide

Static light scattering to characterize membrane proteins in detergent solution

NARCIS (Netherlands)

Slotboom, Dirk Jan; Duurkens, Ria H.; Olieman, Kees; Erkens, Guus B.

2008-01-01

Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium

N-(1-Pyrenyl Maleimide Induces Bak Oligomerization and Mitochondrial Dysfunction in Jurkat Cells

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Pei-Rong Huang

2015-01-01

Full Text Available N-(1-pyrenyl maleimide (NPM is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm, and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

Oligomerization of optically active N-(4-hydroxyphenylmandelamide in the presence of β-cyclodextrin and the minor role of chirality

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Helmut Ritter

2014-10-01

Full Text Available The oxidative oligomerization of a chiral mandelamide derivative (N-(4-hydroxyphenylmandelamide, 1 was performed in the presence of horseradish peroxidase, laccase and N,N'-bis(salicylideneethylenediamine-iron(II to obtain chiral oligophenols 2. The low enantioselectivity of the enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was investigated. In addition, the poor influence of cyclodextrin on the enantioselectivity of enzymatic catalyzed asymmetric enantiomer-differentiating oligomerizations was studied.

40 CFR 721.3100 - Oligomeric silicic acid ester compound with a hy-droxyl-al-kyla-mine.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Oligomeric silicic acid ester compound with a hy-droxyl-al-kyla-mine. 721.3100 Section 721.3100 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3100 Oligomeric silicic acid ester compound with a...

Distinct profile of vascular progenitor attachment to extracellular matrix proteins in cancer patients.

Science.gov (United States)

Labonté, Laura; Li, Yuhua; Addison, Christina L; Brand, Marjorie; Javidnia, Hedyeh; Corsten, Martin; Burns, Kevin; Allan, David S

2012-04-01

Vascular progenitor cells (VPCs) facilitate angiogenesis and initiate vascular repair by homing in on sites of damage and adhering to extracellular matrix (ECM) proteins. VPCs also contribute to tumor angiogenesis and induce angiogenic switching in sites of metastatic cancer. In this study, the binding of attaching cells in VPC clusters that form in vitro on specific ECM proteins was investigated. VPC cluster assays were performed in vitro on ECM proteins enriched in cancer cells and in remodelling tissue. Profiles of VPC clusters from patients with cancer were compared to healthy controls. The role of VEGF and integrin-specific binding of angiogenic attaching cells was addressed. VPC clusters from cancer patients were markedly increased on fibronectin relative to other ECM proteins tested, in contrast to VPC clusters from control subjects, which formed preferentially on laminin. Specific integrin-mediated binding of attaching cells in VPC clusters was matrix protein-dependent. Furthermore, cancer patients had elevated plasma VEGF levels compared to healthy controls and VEGF facilitated preferential VPC cluster formation on fibronectin. Incubating cells from healthy controls with VEGF induced a switch from the 'healthy' VPC binding profile to the profile observed in cancer patients with a marked increase in VPC cluster formation on fibronectin. The ECM proteins laminin and fibronectin support VPC cluster formation via specific integrins on attaching cells and can facilitate patterns of VPC cluster formation that are distinct in cancer patients. Larger studies, however, are needed to gain insight on how tumor angiogenesis may differ from normal repair processes.

Oligomeric structure and chaperone-like activity of Drosophila melanogaster mitochondrial small heat shock protein Hsp22 and arginine mutants in the alpha-crystallin domain.

Science.gov (United States)

Dabbaghizadeh, Afrooz; Finet, Stéphanie; Morrow, Genevieve; Moutaoufik, Mohamed Taha; Tanguay, Robert M

2017-07-01

The structure and chaperone function of DmHsp22WT, a small Hsp of Drosophila melanogaster localized within mitochondria were examined. Mutations of conserved arginine mutants within the alpha-crystallin domain (ACD) domain (R105G, R109G, and R110G) were introduced, and their effects on oligomerization and chaperone function were assessed. Arginine to glycine mutations do not induce significant changes in tryptophan fluorescence, and the mutated proteins form oligomers that are of equal or smaller size than the wild-type protein. They all form oligomer with one single peak as determined by size exclusion chromatography. While all mutants demonstrate the same efficiency as the DmHsp22WT in a DTT-induced insulin aggregation assay, all are more efficient chaperones to prevent aggregation of malate dehydrogenase. Arginine mutants of DmHsp22 are efficient chaperones to retard aggregation of CS and Luc. In summary, this study shows that mutations of arginine to glycine in DmHsp22 ACD induce a number of structural changes, some of which differ from those described in mammalian sHsps. Interestingly, only the R110G-DmHsp22 mutant, and not the expected R109G equivalent to human R140-HspB1, R116-HspB4, and R120-HspB5, showed different structural properties compared with the DmHsp22WT.

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

Science.gov (United States)

Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

2017-08-08

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

NARCIS (Netherlands)

van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

2001-01-01

OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte

Patchwork structure-function analysis of the Sendai virus matrix protein.

Science.gov (United States)

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

In-situ photocrosslinkable nanohybrid elastomer based on polybutadiene/polyhedral oligomeric silsesquioxane

Energy Technology Data Exchange (ETDEWEB)

Mirmohammadi, Seyed Amin, E-mail: mirmohammadi.sa@gmail.com [Department of Polymerization Engineering, Iran Polymer and Petrochemical Institute (IPPI), P. O. Box: 14965/115, Tehran (Iran, Islamic Republic of); Nekoomanesh-Haghighi, Mehdi, E-mail: m.nekoomanesh@ippi.ac.ir [Department of Polymerization Engineering, Iran Polymer and Petrochemical Institute (IPPI), P. O. Box: 14965/115, Tehran (Iran, Islamic Republic of); Mohammadian Gezaz, Somayyeh [Department of Chemical Engineering, Payame Noor University (PNU), P. O. Box: 19395-3697, Tehran (Iran, Islamic Republic of); Bahri-Laleh, Naeimeh [Department of Polymerization Engineering, Iran Polymer and Petrochemical Institute (IPPI), P. O. Box: 14965/115, Tehran (Iran, Islamic Republic of); Atai, Mohammad [Department of Polymer Science, Iran Polymer and Petrochemical Institute (IPPI), P. O. Box: 14965/115, Tehran (Iran, Islamic Republic of)

2016-11-01

Hydroxyl functionalized nano-sized POSS or ethyleneglycol as diol monomers was incorporated to hydroxyl-terminated polybutadiene (HTPBD) chain in the presence of fumaryl chloride as extender. Blue light photocrosslinking system based on camphorquinone (photoinitiator) and dimethylaminoethyl methacrylate (accelerator) was applied to cure these two synthesized fumarate based macromers. Self-crosslinkability of unsaturated macromers and also crosslinking in presence of a reactive diluent were investigated in absence and presence of 1,4-butanediol dimethacrylate, respectively. Finally, photocured samples were characterized by XRD, SEM, equilibrium swelling study, TGA, DMTA, AFM and cell culture. The results showed that incorporation of POSS nanoparticle into the polymer matrix with a perfect distribution and dispersion can enhance thermal stability, mechanical and biocompatibility properties which can prove a good potential of this in-situ photocrosslinkable nanohybrid in medical applications. - Highlights: • Polyhedral oligomeric silsesquioxane was incorporated to HTPBD by fumaryl chloride as extender. • A reactive diluent was used to improve photocrosslinking process of the unsaturated nanohybrid elastomers. • Nanohybrids showed enhanced properties such as thermal stability, mechanical and biocompatibility. • Improved properties were obtained for photocured samples in presence of reactive diluent.

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

Science.gov (United States)

Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemical activation of bituminous coal for hampering oligomerization of organic contaminants.

Science.gov (United States)

Yan, Liang; Sorial, George A

2011-12-15

Activated carbons prepared by KOH activation of bituminous coal were studied for hampering oligomerization of phenolic compounds on its surface. A total of 24 activated carbons with different microporosity and BET surface area were created. The effect of the different variables of the activation process (KOH/bituminous coal ratio, heating temperature, activation time, and flow rate of nitrogen gas) on critical carbon parameters was analyzed. The impact of activated carbon on oligomerization was examined by conducting isotherm experiments at a neutral pH on Carbon(exp) produced with optimal characteristics and granular activated carbon (GAC) F400 for phenol, 2-methylphenol and 2-ethylphenol. These isotherms were collected under anoxic (absence of molecular oxygen) and oxic (presence of molecular oxygen) conditions. The single solute adsorption of phenol, 2-methylphenol and 2-ethylphenol on Carbon(exp) showed no obvious differences between oxic and anoxic environment, which indicated that the Carbon(exp) sample is very effective in hampering the oligomerization of phenolic compounds under oxic conditions. On the other hand, F400, which have lower micropore percentage and BET surface area, significant increases in the adsorptive capacity had been observed when molecular oxygen was present. Copyright © 2011 Elsevier B.V. All rights reserved.

The influence of γ-radiation on biosynthesis of nuclear matrix proteins of hepatic cells of pregnant rats

International Nuclear Information System (INIS)

Mirkhamidova, P.; Shamsutdinova, G.T.; Mirakhmedov, A.K.; Filatova, L.S.; Bul'dyaeva, T.V.; Zbarskij, I.B.

1992-01-01

A study was made of incorporation of 35 S-methionine into nuclear matrix proteins of hepatic cells of pregnant rats and their embryos subjected to single γ-irradiation ( 60 Co, 1 and 2 Gy, 0.0233 Gy/s) on days 3, 13 and 17 of pregrnancy and embryogenesis. On day 21 of pregnancy and embryogenesis a decrease in the rate of incorporation of 35 S-methionine into nuclear matrix proteins was shown to be a function of radiation dose and time of pregnancy and embryogenesis on the moment of exposure

Radiographic progression is associated with resolution of systemic inflammation in patients with axial spondylarthritis treated with tumor necrosis factor α inhibitors

DEFF Research Database (Denmark)

Pedersen, Susanne Juhl; Sørensen, Inge Juul; Lambert, Robert G W

2011-01-01

To investigate the relationship of circulating biomarkers of inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], and YKL-40), angiogenesis (vascular endothelial growth factor), cartilage turnover (C-terminal crosslinking telopeptide of type II collagen [CTX-II], total aggrecan, matrix...... metalloproteinase 3 [MMP-3], and cartilage oligomeric matrix protein [COMP]), and bone turnover (CTX-I and osteocalcin) to inflammation on magnetic resonance imaging (MRI) and radiographic progression in patients with axial spondylarthritis (SpA) beginning tumor necrosis factor α (TNFα) inhibitor therapy....

Radiographic progression is associated with resolution of systemic inflammation in patients with axial spondylarthritis treated with tumor necrosis factor α inhibitors: A study of radiographic progression, inflammation on magnetic resonance imaging, and circulating biomarkers of inflammation

DEFF Research Database (Denmark)

Pedersen, Susanne Juhl; Sørensen, Inge Juul; Lambert, Robert G W

2011-01-01

To investigate the relationship of circulating biomarkers of inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], and YKL-40), angiogenesis (vascular endothelial growth factor), cartilage turnover (C-terminal crosslinking telopeptide of type II collagen [CTX-II], total aggrecan, matrix...... metalloproteinase 3 [MMP-3], and cartilage oligomeric matrix protein [COMP]), and bone turnover (CTX-I and osteocalcin) to inflammation on magnetic resonance imaging (MRI) and radiographic progression in patients with axial spondylarthritis (SpA) beginning tumor necrosis factor a (TNFa) inhibitor therapy....

EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

KAUST Repository

Kandaswamy, Krishna Kumar Umar

2013-01-01

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred. © 2012 Elsevier Ltd.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

Science.gov (United States)

Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

2015-05-22

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Structural Extracellular Matrix Proteins in Urothelial Bladder Cancer

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Andrea Brunner

2007-01-01

Full Text Available The extracellular matrix (ECM plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fi bronectin (FN, tenascin (Tn-C and thrombospondin 1 (TSP1 in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

Science.gov (United States)

Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

2012-01-01

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

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Masahiro Kawahara

2011-01-01

Full Text Available Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD. Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”, and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

Expression of extracellular matrix proteins: tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma

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Monika Ulamec

2015-12-01

Full Text Available Introduction: The interchanged stromal-epithelial relations and altered expression profiles of various extracellular matrix (ECM proteins creates a suitable microenvironment for cancer development and growth. We support the opinion that remodeling of the extracellular matrix (ECM plays an important role in the cancer progression. The aim of this study was to examine the expression of ECM proteins tenascin-C, fibronectin and galectin-3 in prostatic adenocarcinoma. Methods: Glands and surrounding stroma were analyzed in randomly selected specimens from 52 patients with prostate cancer and 28 patients with benign prostatic hyperplasia (BHP. To evaluate the intensity of tenascin-C, fibronectin and galectin-3 expression the percentage of positively immunostained stromal cells was examined.Results: Compared to BPH, stroma of prostatic adenocarcinoma showed statistically significant increase in tenascin-C expression (p<0.001, predominantly around neoplastic glands, while fibronectin (p=0.001 and galectin-3 (p<0.001 expression in the same area was decreased.Conclusions: Our study confirms changes in the expression of ECM proteins of prostate cancer which may have important role in the cancer development.

The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins)

NARCIS (Netherlands)

Faber, Klaas Nico; Haima, Pieter; Gietl, Christine; Harder, Willem; Ab, Geert; Veenhuis, Marten

1994-01-01

Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H.

Homologous ELISA for detection of oligomeric human TNF: properties of the assay.

Science.gov (United States)

Petyovka, N; Lyach, L; Voitenok, N N

1995-10-26

In order to quantify oligomeric human tumor necrosis factor-alpha (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.

Periostin is an extracellular matrix protein required for eruption of incisors in mice

International Nuclear Information System (INIS)

Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira

2006-01-01

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin -/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin -/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

Science.gov (United States)

Ito, Akira; Aoyama, Tomoki; Iijima, Hirotaka; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

2015-05-01

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Inhibitory effect of PTD-OD-HA fusion protein on Bcr-Abl in K562 cells

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Miao GAO

2012-10-01

Full Text Available Objective　To study the transduction dynamics, location of PTD-OD-HA fusion protein and its interaction with Bcr-Abl oncoprotein in K562 cell lines, and explore the influence of PTD-OD-HA fusion protein on oligomerization and tyrosine kinase activity of Bcr-Abl. Methods　PTD-OD-HA fusion protein was labeled with FITC and co-cultured with K562 cells. The transduction efficiency of labeled PTD-OD-HA at different doses and time intervals was observed under fluorescence microscope. The location of labeled PTD-OD-HA fusion protein in K562 cells was detected by confocal microscopy. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was confirmed by coimmunoprecipitation. The phosphorylation of Bcr-Abl oncoprotein was detected by Western blotting. Results　PTD-OD-HA fusion protein labeled with FITC was transduced into K562 cells in a dose- and time-dependent manner. PTD-OD-HA fusion protein was located in the cytoplasm of K562 cells and was consistent with the location of Bcr-Abl oncoprotein. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was proved in K562 cells. This interaction could interrupt the homologous oligomerization of Bcr-Abl oncoprotein and reduce the phosphorylation of Bcr-Abl oncoprotein. Conclusion　PTD-OD-HA fusion protein could be transduced into K562 cells efficiently, inhibit the oligomerization and reduce the phosphorylation of Bcr-Abl oncoprotein.

Polybenzoxazine/Polyhedral Oligomeric Silsesquioxane (POSS Nanocomposites

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Mohamed Gamal Mohamed

2016-06-01

Full Text Available The organic/inorganic hybrid materials from polyhedral oligomeric silsesquioxane (POSS, inorganic nanoparticles and polybenzoxazine (PBZ have received much interesting recently due to their excellent thermal and mechanical properties, flame retardance, low dielectric constant, well-defined inorganic framework at nanosized scale level, and higher performance relative to those of non-hybrid PBZs. This review describes the synthesis, dielectric constants, and thermal, rheological, and mechanical properties of covalently bonded mono- and multifunctionalized benzoxazine POSS hybrids, other functionalized benzoxazine POSS derivatives, and non-covalently (hydrogen bonded benzoxazine POSS composites.

Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

Science.gov (United States)

Weigele, Jochen; Franz-Odendaal, Tamara A; Hilbig, Reinhard

2016-02-01

The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required. © 2015 Wiley Periodicals, Inc.

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

Science.gov (United States)

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

Science.gov (United States)

Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

2016-11-01

Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features. Copyright © 2016. Published by Elsevier Inc.

Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus

Czech Academy of Sciences Publication Activity Database

Doležal, Michal; Zábranský, Aleš; Dostál, Jiří; Vaněk, O.; Brynda, Jiří; Lepšík, Martin; Hadravová, Romana; Pichová, Iva

2016-01-01

Roč. 13, Jan 5 (2016), č. článku 2. ISSN 1742-4690 R&D Projects: GA MŠk LO1302; GA MŠk(CZ) LO1304; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : dimerization * matrix protein * MMTV * molecular dynamics * mouse mammary tumor virus * myristoylation Subject RIV: CE - Biochemistry Impact factor: 3.867, year: 2016 http://retrovirology.biomedcentral.com/articles/10.1186/s12977-015-0235-8

Biochemical and Structural Analysis of Hormone-sensitive Lipase Homolog EstE7: Insight into the Stabilized Dimerization of HSL-Homolog Proteins

International Nuclear Information System (INIS)

Nam, Ki Hyun; Park, Sung Ha; Lee, Won Ho; Hwang, Kwang Yeon

2010-01-01

Hormone sensitive lipase (HSL) plays a major role in energy homeostasis and lipid metabolism. Several crystal structures of HSL-homolog proteins have been identified, which has led to a better understanding of its molecular function. HSLhomolog proteins exit as both monomer and dimer, but the biochemical and structural basis for such oligomeric states has not been successfully elucidated. Therefore, we determined the crystal structure of HSL-homolog protein EstE7 from a metagenome library at 2.2 A resolution and characterized the oligomeric states of EstE7 both structurally and biochemically. EstE7 protein prefers the dimeric state in solution, which is supported by its higher enzymatic activity in the dimeric state. In the crystal form, EstE7 protein shows two-types of dimeric interface. Specifically, dimerization via the external β8-strand occurred through tight association between two pseudosymmetric folds via salt bridges, hydrogen bonds and van der Waals interactions. This dimer formation was similar to that of other HSL-homolog protein structures such as AFEST, BEFA, and EstE1. We anticipate that our results will provide insight into the oligomeric state of HSLhomolog proteins

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

International Nuclear Information System (INIS)

Heremans, A.; Cassiman, J.J.; Van den Berghe, H.; David, G.

1988-01-01

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

Influence of secondary structure on in-source decay of protein in matrix-assisted laser desorption/ionization mass spectrometry.

Science.gov (United States)

Takayama, Mitsuo; Osaka, Issey; Sakakura, Motoshi

2012-01-01

The susceptibility of the N-Cα bond of the peptide backbone to specific cleavage by in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was studied from the standpoint of the secondary structure of three proteins. A naphthalene derivative, 5-amino-1-naphtol (5,1-ANL), was used as the matrix. The resulting c'-ions, which originate from the cleavage at N-Cα bonds in flexible secondary structures such as turn and bend, and are free from intra-molecular hydrogen-bonded α-helix structure, gave relatively intense peaks. Furthermore, ISD spectra of the proteins showed that the N-Cα bonds of specific amino acid residues, namely Gly-Xxx, Xxx-Asp, and Xxx-Asn, were more susceptible to MALDI-ISD than other amino acid residues. This is in agreement with the observation that Gly, Asp and Asn residues usually located in turns, rather than α-helix. The results obtained indicate that protein molecules embedded into the matrix crystal in the MALDI experiments maintain their secondary structures as determined by X-ray crystallography, and that MALDI-ISD has the capability for providing information concerning the secondary structure of protein.

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

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Yao E Wang

2010-11-01

Full Text Available Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4 pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS and the leucine-rich nuclear export signal (NES found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Enhance and Maintain Chondrogenesis of Synovial Fibroblasts by Cartilage Extracellular Matrix Protein Matrilins

Science.gov (United States)

Pei, Ming; Luo, Junming; Chen, Qian

2008-01-01

Summary Objective Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN) 1 and 3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Methods Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time RT-PCR, while the protein levels of Col II and matrilins were determined by western blot analysis. Results SFBs underwent chondrogenesis after incubation with TGF-β1 for three days; however, this process was attenuated during the subsequent incubation period. Expression of a MATN1 or 3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of glycosaminoglycans, and stimulated expression of chondrogenic markers. Conclusion Our findings suggest a novel function for MATN1 and 3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-β. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation. PMID:18282772

Enhancing and maintaining chondrogenesis of synovial fibroblasts by cartilage extracellular matrix protein matrilins.

Science.gov (United States)

Pei, M; Luo, J; Chen, Q

2008-09-01

Cartilage-specific extracellular matrix (ECM) proteins have been proposed to play key roles in modulating cellular phenotypes during chondrogenesis of mesenchymal stem cells. Matrilin (MATN)1 and MATN3 are among the most up-regulated ECM proteins during chondrogenesis. The aim of this study was to analyze their roles in chondrogenesis of mesenchymal fibroblasts from synovium. Primary synovial fibroblasts (SFBs) were purified from porcine synovium and incubated in pellet culture for 18 days. Chondrogenesis of SFB was analyzed by histological staining with safranin-O/fast green, and by quantifying glycosaminoglycans (GAG) with dimethylmethylene blue assay. The mRNA levels of chondrogenic markers including collagen II, aggrecan, and Sox 9 were quantified by real-time reverse transcription polymerase chain reaction, while the protein levels of Col II and MATNs were determined by western blot analysis. SFBs underwent chondrogenesis after incubation with transforming growth factor-beta1 (TGF-beta1) for 3 days; however, this process was attenuated during the subsequent incubation period. Expression of a Matn1 or Matn3 cDNA maintained and further enhanced chondrogenesis of SFBs as shown by increased cartilaginous matrix areas, elevated amount of GAG, and stimulated expression of chondrogenic markers. Our findings suggest a novel function for MATN1 and MATN3 to maintain and enhance chondrogenesis of mesenchymal fibroblasts initiated by TGF-beta. Our results also support a critical role of cartilage-specific ECM proteins to modulate cellular phenotypes in the microenvironment during chondrogenic differentiation.

Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

Science.gov (United States)

Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

2015-10-14

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

International Nuclear Information System (INIS)

Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro; Yamaguchi, Kazuo; Garcia, Andres J

2011-01-01

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII 7-10 ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Energy Technology Data Exchange (ETDEWEB)

Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro [World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science - NIMS (Japan); Yamaguchi, Kazuo [Department of Chemistry, Faculty of Science and Research Institute for Photofunctionalized Materials, Kanagawa University (Japan); Garcia, Andres J, E-mail: NAKANISHI.Jun@nims.go.jp [Institute for Bioengineering and Bioscience, Woodruff School of Mechanical Engineering, Georgia Institute of Technology (United States)

2011-08-15

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG{sub 7}). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni{sup 2+}-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG{sub 7} underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII{sub 7-10}) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII{sub 7-10} was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

Science.gov (United States)

Watson, Steve P

2009-01-01

The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

Thermo- and pH-responsive polymer brushes-grafted gigaporous polystyrene microspheres as a high-speed protein chromatography matrix.

Science.gov (United States)

Qu, Jian-Bo; Xu, Yu-Liang; Liu, Jun-Yi; Zeng, Jing-Bin; Chen, Yan-Li; Zhou, Wei-Qing; Liu, Jian-Guo

2016-04-08

Dual thermo- and pH-responsive chromatography has been proposed using poly(N-isopropylacrylamide-co-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAM-co-BMA-co-DMAPAAM)) brushes grafted gigaporous polystyrene microspheres (GPM) as matrix. Atom transfer radical polymerization (ATRP) initiator was first coupled onto GPM through Friedel-Crafts acylation with 2-bromoisobutyryl bromide. The dual-responsive polymer brushes were then grafted onto GPM via surface-initiated ATRP. The surface composition, gigaporous structure, protein adsorption and dual-responsive chromatographic properties of the matrix (GPM-P(NIPAM-co-BMA-co-DMAPAAM) were characterized in detail. Results showed that GPM were successfully grafted with thermoresponsive cationic polymer brushes and that the gigaporous structure was well maintained. A column packed with GPM-P(NIPAM-co-BMA-co-DMAPAAM presented low backpressure, good permeability and appreciable thermo-responsibility. By changing pH of the mobile phase and temperature of the column in turn, the column can separate three model proteins at the mobile phase velocity up to 2528cmh(-1). A separation mechanism of this matrix was also proposed. All results indicate that the dual thermo- and pH-responsive chromatography matrix has great potentials in 'green' high-speed protein chromatography. Copyright © 2016 Elsevier B.V. All rights reserved.

β-TCP/HA with or without enamel matrix proteins for maxillary sinus floor augmentation

DEFF Research Database (Denmark)

Nery, James Carlos; Pereira, Luís Antônio Violin Dias; Guimarães, George Furtado

2017-01-01

BACKGROUND: It is still unclear whether enamel matrix proteins (EMD) as adjunct to bone grafting enhance bone healing. This study compared histomorphometrically maxillary sinus floor augmentation (MSFA) with β-TCP/HA in combination with or without EMD in humans. METHODS: In ten systemically healthy...

Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

Science.gov (United States)

Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

2016-01-01

The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

Understanding curcumin-induced modulation of protein aggregation.

Science.gov (United States)

Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P

2017-07-01

Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein aggregates and novel presenilin gene variants in idiopathic dilated cardiomyopathy.

Science.gov (United States)

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E; del Monte, Federica

2010-03-16

Heart failure is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as idiopathic dilated cardiomyopathy (iDCM), the origin of heart failure is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of beta-amyloid impair cell function and lead to cell death. We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients, pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca(2+) homeostasis. Additionally, we have identified 2 new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 coimmunoprecipitates with SERCA2a. On the basis of these findings, we propose that 2 mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca(2+) handling and a direct effect of PSEN1 sequence variants on excitation-contraction coupling protein function.

Antibodies against alpha-synuclein reduce oligomerization in living cells.

Directory of Open Access Journals (Sweden)

Thomas Näsström

Full Text Available Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

International Nuclear Information System (INIS)

Sung, Nak-Yun; Choi, Jong-il

2015-01-01

Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60 cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants - Highlights: • Demineralized bone matrix (DBM) was gamma-irradiated for sterilization. • Irradiated DBM had higher alkaline phosphatase and osteocalcin production. • It was reasoned the more released bone morphogenetic proteins by irradiation. • This result supports the application of radiation sterilization for bone implants

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

Science.gov (United States)

Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

2016-11-01

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone.

Directory of Open Access Journals (Sweden)

Jason Trigg

Full Text Available The alpha-helical coiled coil can adopt a variety of topologies, among the most common of which are parallel and antiparallel dimers and trimers. We present Multicoil2, an algorithm that predicts both the location and oligomerization state (two versus three helices of coiled coils in protein sequences. Multicoil2 combines the pairwise correlations of the previous Multicoil method with the flexibility of Hidden Markov Models (HMMs in a Markov Random Field (MRF. The resulting algorithm integrates sequence features, including pairwise interactions, through multinomial logistic regression to devise an optimized scoring function for distinguishing dimer, trimer and non-coiled-coil oligomerization states; this scoring function is used to produce Markov Random Field potentials that incorporate pairwise correlations localized in sequence. Multicoil2 significantly improves both coiled-coil detection and dimer versus trimer state prediction over the original Multicoil algorithm retrained on a newly-constructed database of coiled-coil sequences. The new database, comprised of 2,105 sequences containing 124,088 residues, includes reliable structural annotations based on experimental data in the literature. Notably, the enhanced performance of Multicoil2 is evident when tested in stringent leave-family-out cross-validation on the new database, reflecting expected performance on challenging new prediction targets that have minimal sequence similarity to known coiled-coil families. The Multicoil2 program and training database are available for download from http://multicoil2.csail.mit.edu.

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

Science.gov (United States)

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

A cell-free assay to determine the stoichiometry of plasma membrane proteins.

Science.gov (United States)

Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

2013-04-01

Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

Protein-transitions in and out of the dough matrix in wheat flour mixing.

Science.gov (United States)

Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

2017-02-15

Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Directory of Open Access Journals (Sweden)

Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

2011-01-01

Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Ion Channel Conformation and Oligomerization Assessment by Site-Directed Spin Labeling and Pulsed-EPR.

Science.gov (United States)

Pliotas, Christos

2017-01-01

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail. © 2017 Elsevier Inc. All rights reserved.

Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

Energy Technology Data Exchange (ETDEWEB)

Rusen, L. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH, Magurele, Bucharest (Romania); Mitu, B.; Filipescu, M.; Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania)

2013-08-01

Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm{sup −2}. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

International Nuclear Information System (INIS)

Gualdrón-López, Melisa; Michels, Paul A.M.

2013-01-01

Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M r of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M r of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and 35 S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed

Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

Energy Technology Data Exchange (ETDEWEB)

Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

2013-02-01

Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

Glycation of extracellular matrix proteins and its role in atherosclerosis 

Directory of Open Access Journals (Sweden)

Aleksandra Kuzan

2012-10-01

Full Text Available Glycation consists in formation of advanced glycation end-products (AGE during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins’ expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such aspyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models.

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Anat Frydman-Marom

Full Text Available An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ play a key role in Alzheimer's disease (AD pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt, which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.

N-way FRET microscopy of multiple protein-protein interactions in live cells.

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Adam D Hoppe

Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

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Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

2015-01-01

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions

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Gupta, Kalpana; Ott, David; Hope, Thomas J.; Siliciano, Robert F.; Boeke, Jef D.

2000-01-01

Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55Gag and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4+ T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55Gag and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport. PMID:11090181

AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

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Xia Luo

2015-01-01

Full Text Available Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f. Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1 whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1. In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix.

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Yun, Jing-Ping; Chew, Eng Ching; Liew, Choong-Tsek; Chan, John Y H; Jin, Mei-Lin; Ding, Ming-Xiao; Fai, Yam Hin; Li, H K Richard; Liang, Xiao-Man; Wu, Qiu-Liang

2003-12-15

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix. Copyright 2003 Wiley-Liss, Inc.

The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro.

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Audrey Bagnaud-Baule

Full Text Available Each year, during winter months, human Metapneumovirus (hMPV is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.

NMR structure of the myristylated feline immunodeficiency virus matrix protein.

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Brown, Lola A; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G; Kuo, Lillian; Freed, Eric O; Summers, Michael F

2015-04-30

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag's N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

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Lola A. Brown

2015-04-01

Full Text Available Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2 is mediated by Gag’s N-terminally myristylated matrix (MA domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV, a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S. These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly.

An Investigation of Chemical Landscapes in Aqueous Electrosprays by Tracking Oligomerization of Isoprene

KAUST Repository

Junior, Adair Gallo

2017-12-01

Electrospray ionization mass spectrometry (ESIMS) is widely used to characterize neutral and ionic species in solvents. Typically, electrical, thermal, and pneumatic potentials are applied to create electrosprays from which charged ionic species are ejected for downstream analysis by mass spectrometry. Most recently, ESIMS has been exploited to investigate ambient proton transfer reactions at air-water interfaces in real time. We assessed the validity of these experiments via complementary laboratory experiments. Specifically, we characterized the products of two reaction scenarios via ESIMS and proton nuclear magnetic resonance (1H-NMR): (i) emulsions of pH-adjusted water and isoprene (C5H8) that were mechanically agitated, and (ii) electrosprays of pH-adjusted water that were collided with gas-phase isoprene. Our experiments unambiguously demonstrate that, while isoprene does not oligomerize in emulsions, it does undergo protonation and oligomerization in electrosprays, both with and without pH-adjusted water, confirming that C-C bonds form along myriad high-energy pathways during electrospray ionization. We also compared our experimental results with some quantum mechanics simulations of isoprene molecules interacting with hydronium at different hydration levels (gas versus liquid phase). In agreement with our experiments, the kinetic barriers to protonation and oligomerization of isoprene were inaccessible under ambient conditions. Rather, the gas-phase chemistries during electrospray ionization drove the oligomerization of isoprene. Therefore, we consider that ESIMS could induce artifacts in interfacial reactions. These findings warrant a reassessment of previous reports on tracking chemistries under ambient conditions at liquid-vapor interfaces via ESIMS. Further, we took some high-speed images of electrosprays where it was possible to observe the main characteristics of the phenomena, i.e. Taylor cone, charge separation, and Coulomb fission. Finally, we took

The application of an enamel matrix protein derivative (Emdogain) in regenerative periodontal therapy: a review.

NARCIS (Netherlands)

Sculean, A.; Schwarz, F.; Becker, J.; Brecx, M.

2007-01-01

Regenerative periodontal therapy aims at reconstitution of the lost periodontal structures such as new formation of root cementum, periodontal ligament and alveolar bone. Findings from basic research indicate that enamel matrix protein derivative (EMD) has a key role in periodontal wound healing.

Catalysis of the Oligomerization of O-Phospho-Serine, Aspartic Acid, or Glutamic Acid by Cationic Micelles

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Bohler, Christof; Hill, Aubrey R., Jr.; Orgel, Leslie E.

1996-01-01

Treatment of relatively concentrated aqueous solutions of 0-phospho-serine (50 mM), aspartic acid (100 mM) or glutamic acid (100 mM) with carbonyldiimidazole leads to the formation of an activated intermediate that oligomerizes efficiently. When the concentration of amino acid is reduced tenfold, few long oligomers can be detected. Positively-charged cetyltrimethyl ammonium bromide micelles concentrate the negatively-charged activated intermediates of the amino acids at their surfaces and catalyze efficient oligomerization even from dilute solutions.

Glycerol-Induced Aggregation of the Oligomeric L-Asparaginase II from E. coli Monitored with ATR-FTIR

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Koba Adeishvili

2001-06-01

Full Text Available Abstract: In this paper attenuated total reflectance Fourier transform infrared spectroscopy has been employed for the study of the structural composition of aggregates of the oligomeric L-asparaginase II from E.coli formed in the presence of glycerol after the induction of refolding of the protein. Apart from the perfect coincidence of the secondary structure composition of EcA2 as determined by FTIR and crystallography [1], it has also been shown that secondary structure of protein in asparaginase deposits is similar to that of the native conformation: 20.7% extended, 22.3% disordered, 31.4% helix and 25.6% turn/bend/β sheet. Certain structural similarities in the range of experimental error was observed for all three protein deposits presented in this paper, indicating a common structural basis for the composition of this types of aggregates. It is concluded that in the constitution of such precipitates, a partially folded (molten globule like state(s is involved, and its stabilisation is a key factor leading to the aggregation. The results presented in this paper might serve to be a good explanation and an excellent basis for the fundamental theory of protein (oligomers precipitation by osmotic substances.

The role of surface electrostatics on the stability, function and regulation of human cystathionine β-synthase, a complex multidomain and oligomeric protein.

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Pey, Angel L; Majtan, Tomas; Kraus, Jan P

2014-09-01

Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

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Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

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Xiuli Lu

2013-02-01

Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

Hyriopsis cumingii Hic52-A novel nacreous layer matrix protein with a collagen-like structure.

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Liu, Xiaojun; Pu, Jingwen; Zeng, Shimei; Jin, Can; Dong, Shaojian; Li, Jiale

2017-09-01

Nacre is a product of a precisely regulated biomineralization process and a major contributor to the luster of pearls. Nacre is composed of calcium carbonate and an organic matrix of proteins that is secreted from mollusc mantle tissue and is exclusively associated with shell formation. In this study, hic52, a novel matrix protein gene from mantle of Hyriopsis cumingii, was cloned and functionally analyzed. The full-length cDNA of hic52 encoded 542 amino acids and contained a signal peptide of 18 amino acids. Excluding the signal peptide, the theoretical molecular mass of the polypeptide was 52.2kDa. The predicted isoelectric point was 10.37, indicating a basic shell protein. The amino acid sequence of hic52 featured high proportion of Gly (28.8%) and Gln (12.4%) residues. The predicted tertiary structure was characterized as having similarities to collagen I, alpha 1 and alpha 2 in the structure. The polypeptide sequence shared no homology with collagen. The hic52 expression pattern by quantitative real-time PCR and in situ hybridization exhibits at the dorsal epithelial cells of the mantle. Expression increased during the stages of pearl sac development. The data showed that hic52 is probably a framework shell protein that mediates and controls the nacreous biomineralization process. Copyright © 2017 Elsevier B.V. All rights reserved.

Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice

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Fernanda S. Oliveira

2012-01-01

Full Text Available Nucleotide-binding oligomerization domain proteins (NODs are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

International Nuclear Information System (INIS)

Nieznanski, Krzysztof; Podlubnaya, Zoya A.; Nieznanska, Hanna

2006-01-01

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for First time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of ∼50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers

Protic ionic liquids based on the dimeric and oligomeric anions: [(AcO)xH(x-1)]-.

Science.gov (United States)

Johansson, K M; Izgorodina, E I; Forsyth, M; MacFarlane, D R; Seddon, K R

2008-05-28

We describe a fluidity and conductivity study as a function of composition in N-methylpyrrolidine-acetic acid mixtures. The simple 1 : 1 acid-base mixture appears to form an ionic liquid, but its degree of ionicity is quite low and such liquids are better thought of as poorly dissociated mixtures of acid and base. The composition consisting of 3 moles acetic acid and 1 mole N-methylpyrrolidine is shown to form the highest ionicity mixture in this binary due to the presence of oligomeric anionic species [(AcO)(x)H(x-1)](-) stabilised by hydrogen bonds. These oligomeric species, being weaker bases than the acetate anion, shift the proton transfer equilibrium towards formation of ionic species, thus generating a higher degree of ionicity than is present at the 1 : 1 composition. A Walden plot analysis, thermogravimetric behaviour and proton NMR data, as well as ab initio calculations of the oligomeric species, all support this conclusion.

3DSwap: Curated knowledgebase of proteins involved in 3D domain swapping

KAUST Repository

Shameer, Khader; Shingate, Prashant N.; Manjunath, S. C. P.; Karthika, M.; Pugalenthi, Ganesan; Sowdhamini, Ramanathan

2011-01-01

structures in oligomeric conformation. Protein structures in swapped conformations perform diverse functional roles and are also associated with deposition diseases in humans. We have performed in-depth literature curation and structural bioinformatics

Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

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Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

2015-08-28

The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

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Emilie eGauliard

2015-02-01

Full Text Available Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins. Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization.

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Tobias Maisel

Full Text Available CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk, preparations of CoxD revealed a mean K(M value of 0.69±0.14 mM ATP and an apparent V(max value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer, preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.

Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards

DEFF Research Database (Denmark)

Mirgorodskaya, O A; Kozmin, Y P; Titov, M I

2000-01-01

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for...... inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.......A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards...

Protein Aggregates and Novel Presenilin Gene Variants in Idiopathic Dilated Cardiomyopathy

Science.gov (United States)

Gianni, Davide; Li, Airong; Tesco, Giuseppina; McKay, Kenneth M.; Moore, John; Raygor, Kunal; Rota, Marcello; Gwathmey, Judith K; Dec, G William; Aretz, Thomas; Leri, Annarosa; Semigran, Marc J; Anversa, Piero; Macgillivray, Thomas E; Tanzi, Rudolph E.; Monte, Federica del

2010-01-01

Background Heart failure (HF) is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as Idiopathic Dilated Cardiomyopathy (iDCM), the origin of HF is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of β-amyloid impair cell function and lead to cell death. Methods and Results We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca2+ homeostasis. Additionally, we have identified two new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 co-immunoprecipitates with SERCA2a. Conclusions Based on these findings we propose that two mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca2+ handling and a direct effect of PSEN1 sequence variants on EC-coupling protein function. PMID:20194882

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

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Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

Comparative proteomics of matrix fractions between pimpled and normal chicken eggshells.

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Liu, Zhangguo; Song, Lingzi; Lu, Lizhi; Zhang, Xianfu; Zhang, Fuming; Wang, Kehua; Linhardt, Robert J

2017-09-07

Eggshell matrix can be dissociated into three matrix fractions: acid-insoluble matrix (M1), water-insoluble matrix (M2) and acid-water facultative-soluble matrix (M3). Matrix fractions from pimpled and normal eggshells were compared using label-free proteomic method to understand the differences among three matrix fractions and the proteins involved with eggshell quality. A total of 738 and 600 proteins were identified in the pimpled and normal calcified eggshells, respectively. Both eggshells showed a combined proteomic inventory of 769 proteins. In the same type of eggshell, a high similarity was present in the proteomes of three matrix fractions. These triply overlapped common proteins formed the predominant contributor to proteomic abundance in the matrix fractions. In each matrix fraction and between both eggshell models, normal and pimpled eggshells, a majority of the proteomes of the fractions were commonly observed. Forty-two common major proteins (iBAQ-derived abundance ≥0.095% of proteomic abundance) were identified throughout the three matrix fractions and these proteins might act as backbone constituents in chicken eggshell matrix. Finally, using 1.75-fold as up-regulated and using 0.57-fold as down-regulated cutoff values, twenty-five differential major proteins were screened and they all negatively influence and none showed any effect on eggshell quality. Overall, we uncovered the characteristics of proteomics of three eggshell matrix fractions and identified candidate proteins influencing eggshell quality. The next research on differential proteins will uncover the potential mechanisms underlying how proteins affect eggshell quality. It was reported that the proteins in an eggshell can be divided into insoluble and soluble proteins. The insoluble proteins are thought to be an inter-mineral matrix and acts as a structural framework, while the soluble proteins are thought as intra-mineral matrix that are embedded within the crystal during

One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

Science.gov (United States)

Lock, Antonia; Forfar, Rachel; Weston, Cathryn; Bowsher, Leo; Upton, Graham J G; Reynolds, Christopher A; Ladds, Graham; Dixon, Ann M

2014-12-01

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs. Copyright © 2014. Published by Elsevier B.V.

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

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Shishkov, Alexander; Bogacheva, Elena; Fedorova, Natalia; Ksenofontov, Alexander; Badun, Gennadii; Radyukhin, Victor; Lukashina, Elena; Serebryakova, Marina; Dolgov, Alexey; Chulichkov, Alexey; Dobrov, Evgeny; Baratova, Lyudmila

2011-12-01

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell. © 2011 The Authors Journal compilation © 2011 FEBS.

Polyhedral Oligomeric Silsesquioxane (POSS)-Containing Polymer Nanocomposites

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Ayandele, Ebunoluwa; Sarkar, Biswajit; Alexandridis, Paschalis

2012-01-01

Hybrid materials with superior structural and functional properties can be obtained by incorporating nanofillers into polymer matrices. Polyhedral oligomeric silsesquioxane (POSS) nanoparticles have attracted much attention recently due to their nanometer size, the ease of which these particles can be incorporated into polymeric materials and the unique capability to reinforce polymers. We review here the state of POSS-containing polymer nanocomposites. We discuss the influence of the incorporation of POSS into polymer matrices via chemical cross-linking or physical blending on the structure of nanocomposites, as affected by surface functional groups, and the POSS concentration. PMID:28348318

Identification of a Novel Dentin Matrix Protein-1 (DMP-1) Mutation and Dental Anomalies in a Kindred with Autosomal Recessive Hypophosphatemia

OpenAIRE

Turan, Serap; Aydin, Cumhur; Bereket, Abdullah; Akcay, Teoman; Güran, Tülay; Yaralioglu, Betul Akmen; Bastepe, Murat; Jüppner, Harald

2009-01-01

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we report a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowin...

Circulating matrix gamma-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome

NARCIS (Netherlands)

Cranenburg, E. C. M.; van Spaendonck-Zwarts, K. Y.; Bonafe, L.; Crettol, L. Mittaz; Rodiger, L. A.; Dikkers, F. G.; van Essen, A. J.; Superti-Furga, A.; Alexandrakis, E.; Vermeer, C.; Schurgers, L. J.; Laverman, G. D.

Background and objectives: Matrix gamma-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an

Circulating matrix gamma-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome

NARCIS (Netherlands)

Cranenburg, E. C. M.; van Spaendonck-Zwarts, K. Y.; Bonafe, L.; Mittaz Crettol, L.; Rödiger, L. A.; Dikkers, F. G.; van Essen, A. J.; Superti-Furga, A.; Alexandrakis, E.; Vermeer, C.; Schurgers, L. J.; Laverman, G. D.

2011-01-01

Background and objectives: Matrix gamma-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an

In-depth, high-accuracy proteomics of sea urchin tooth organic matrix

Directory of Open Access Journals (Sweden)

Mann Matthias

2008-12-01

Full Text Available Abstract Background The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis. Results We identified 138 proteins in the matrix of tooth powder. Only 56 of these proteins were previously identified in the matrices of test (shell and spine. Among the novel components was an interesting group of five proteins containing alanine- and proline-rich neutral or basic motifs separated by acidic glycine-rich motifs. In addition, four of the five proteins contained either one or two predicted Kazal protease inhibitor domains. The major components of tooth matrix were however largely identical to the set of spicule matrix proteins and MSP130-related proteins identified in test (shell and spine matrix. Comparison of the matrices of crushed teeth to intact teeth revealed a marked dilution of known intracrystalline matrix proteins and a concomitant increase in some intracellular proteins. Conclusion This report presents the most comprehensive list of sea urchin tooth matrix proteins available at present. The complex mixture of proteins identified may reflect many different aspects of the mineralization process. A comparison between intact tooth matrix, presumably containing odontoblast remnants, and crushed tooth matrix served to differentiate between matrix components and possible contributions of cellular remnants. Because LC-MS/MS-based methods directly

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

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Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage

DEFF Research Database (Denmark)

Roy, Roopali; Wewer, Ulla M; Zurakowski, David

2004-01-01

-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-k......Da band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen...... or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme...

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

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Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

Role of redox environment on the oligomerization of higher molecular weight adiponectin

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Nuñez Martha

2011-05-01

Full Text Available Abstract Background Adiponectin is an adipocyte-secreted hormone with insulin-sensitizing and anti-inflammatory actions. The assembly of trimeric, hexameric, and higher molecular weight (HMW species of adiponectin is a topic of significant interest because physiological actions of adiponectin are oligomer-specific. In addition, adiponectin assembly is an example of oxidative oligomerization of multi-subunit protein complexes in endoplasmic reticulum (ER. Results We previously reported that trimers assemble into HMW adiponectin via intermediates stabilized by disulfide bonds, and complete oxidation of available cysteines locks adiponectin in hexameric conformation. In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers. Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species. Increased ratios of HMW to hexameric adiponectin could be achieved rapidly under oxidizing conditions by promoting disulfide rearrangement. Conclusions Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach

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Daniel V. Oliveira

2012-01-01

Full Text Available Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM. We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2 were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

In vitro folding of inclusion body proteins.

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Rudolph, R; Lilie, H

1996-01-01

Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

International Nuclear Information System (INIS)

Farhat, Walid A; Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman; Sherman, Christopher; Derwin, Kathleen

2008-01-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

Science.gov (United States)

Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

Energy Technology Data Exchange (ETDEWEB)

Farhat, Walid A [Department of Surgery, Division of Urology, University of Toronto and Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Chen Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Yeger, Herman [Department of Developmental and Stem Cell Biology, Research Institute, Hospital for Sick Children, Toronto, ON M5G 1X8 (Canada); Sherman, Christopher [Department of Anatomic Pathology, Sunnybrook and Women' s College Health Sciences Centre, Toronto, ON (Canada); Derwin, Kathleen [Department of Biomedical Engineering, Lerner Research Institute and Orthopaedic Research Center, Cleveland Clinic Foundation, Cleveland, OH (United States)], E-mail: walid.farhat@sickkids.ca

2008-06-01

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

Science.gov (United States)

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

DEFF Research Database (Denmark)

Callesen, Anne K; Christensen, René Depont; Madsen, Jonna S

2008-01-01

for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom......Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum......-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard...

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

Science.gov (United States)

Tseng, Boo Shan; Reichhardt, Courtney; Merrihew, Gennifer E; Araujo-Hernandez, Sophia A; Harrison, Joe J; MacCoss, Michael J; Parsek, Matthew R

2018-04-10

Pseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response. IMPORTANCE Proteins associated with the extracellular matrix of bacterial aggregates called biofilms have long been suggested to provide many important functions to the community. To date, however, only proteins that provide structural roles have been described, and few matrix-associated proteins have been identified. We developed a method to identify matrix proteins and characterized one. We show that this protein, when associated with the biofilm matrix, can inhibit a bactericidal enzyme produced by the immune system during infection and protect biofilm cells from death induced by the enzyme. This may represent a novel mechanism of protection for biofilms, further increasing their tolerance against the immune response. Together, our results are the first to show a nonstructural function for a confirmed matrix

EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

KAUST Repository

Kandaswamy, Krishna Kumar Umar; Ganesan, Pugalenthi; Kalies, Kai Uwe; Hartmann, Enno; Martinetz, Thomas M.

2013-01-01

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan

microRNA as a potential vector for the propagation of robustness in protein expression and oscillatory dynamics within a ceRNA network.

Directory of Open Access Journals (Sweden)

Claude Gérard

Full Text Available microRNAs (miRNAs are small noncoding RNAs that are important post-transcriptional regulators of gene expression. miRNAs can induce thresholds in protein synthesis. Such thresholds in protein output can be also achieved by oligomerization of transcription factors (TF for the control of gene expression. First, we propose a minimal model for protein expression regulated by miRNA and by oligomerization of TF. We show that miRNA and oligomerization of TF generate a buffer, which increases the robustness of protein output towards molecular noise as well as towards random variation of kinetics parameters. Next, we extend the model by considering that the same miRNA can bind to multiple messenger RNAs, which accounts for the dynamics of a minimal competing endogenous RNAs (ceRNAs network. The model shows that, through common miRNA regulation, TF can control the expression of all proteins formed by the ceRNA network, even if it drives the expression of only one gene in the network. The model further suggests that the threshold in protein synthesis mediated by the oligomerization of TF can be propagated to the other genes, which can increase the robustness of the expression of all genes in such ceRNA network. Furthermore, we show that a miRNA could increase the time delay of a "Goodwin-like" oscillator model, which may favor the occurrence of oscillations of large amplitude. This result predicts important roles of miRNAs in the control of the molecular mechanisms leading to the emergence of biological rhythms. Moreover, a model for the latter oscillator embedded in a ceRNA network indicates that the oscillatory behavior can be propagated, via the shared miRNA, to all proteins formed by such ceRNA network. Thus, by means of computational models, we show that miRNAs could act as vectors allowing the propagation of robustness in protein synthesis as well as oscillatory behaviors within ceRNA networks.

Detection of HOCl-mediated protein oxidation products in the extracellular matrix of human atherosclerotic plaques

DEFF Research Database (Denmark)

Woods, Alan A; Linton, Stuart M; Davies, Michael Jonathan

2003-01-01

Oxidation is believed to play a role in atherosclerosis. Oxidized lipids, sterols and proteins have been detected in early, intermediate and advanced human lesions at elevated levels. The spectrum of oxidized side-chain products detected on proteins from homogenates of advanced human lesions has...... been interpreted in terms of the occurrence of two oxidative mechanisms, one involving oxygen-derived radicals catalysed by trace transition metal ions, and a second involving chlorinating species (HOCl or Cl2), generated by the haem enzyme myeloperoxidase (MPO). As MPO is released extracellularly...... for 83-96% of the total oxidized protein side-chain products detected in these plaques. Oxidation of matrix components extracted from healthy artery tissue, and model proteins, with reagent HOCl is shown to give rise to a similar pattern of products to those detected in advanced human lesions...

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

Science.gov (United States)

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

Oligomerization of a Glucagon-like Peptide 1 Analog: Bridging Experiment and Simulations

DEFF Research Database (Denmark)

Frederiksen, Tine Maja; Sønderby, Pernille; Ryberg, Line A.

2015-01-01

The glucagon-like peptide 1 (GLP-1) analog, liraglutide, is a GLP-1 agonist and is used in the treatment of type-2 diabetes mellitus and obesity. From a pharmaceutical perspective, it is important to know the oligomerization state of liraglutide with respect to stability. Compared to GLP-1...

Phrase Mining of Textual Data to Analyze Extracellular Matrix Protein Patterns Across Cardiovascular Disease.

Science.gov (United States)

Liem, David Alexandre; Murali, Sanjana; Sigdel, Dibakar; Shi, Yu; Wang, Xuan; Shen, Jiaming; Choi, Howard; Caufield, J Harry; Wang, Wei; Ping, Peipei; Han, Jiawei

2018-05-18

Extracellular matrix (ECM) proteins have been shown to play important roles regulating multiple biological processes in an array of organ systems, including the cardiovascular system. By using a novel bioinformatics text-mining tool, we studied six categories of cardiovascular disease (CVD), namely ischemic heart disease (IHD), cardiomyopathies (CM), cerebrovascular accident (CVA), congenital heart disease (CHD), arrhythmias (ARR), and valve disease (VD), anticipating novel ECM protein-disease and protein-protein relationships hidden within vast quantities of textual data. We conducted a phrase-mining analysis, delineating the relationships of 709 ECM proteins with the six groups of CVDs reported in 1,099,254 abstracts. The technology pipeline known as Context-aware Semantic Online Analytical Processing (CaseOLAP) was applied to semantically rank the association of proteins to each and all six CVDs, performing analyses to quantify each protein-disease relationship. We performed principal component analysis and hierarchical clustering of the data, where each protein is visualized as a six dimensional vector. We found that ECM proteins display variable degrees of association with the six CVDs; certain CVDs share groups of associated proteins whereas others have divergent protein associations. We identified 82 ECM proteins sharing associations with all six CVDs. Our bioinformatics analysis ascribed distinct ECM pathways (via Reactome) from this subset of proteins, namely insulin-like growth factor regulation and interleukin-4 and interleukin-13 signaling, suggesting their contribution to the pathogenesis of all six CVDs. Finally, we performed hierarchical clustering analysis and identified protein clusters associated with a targeted CVD; analyses revealed unexpected insights underlying ECM-pathogenesis of CVDs.

Peroxisomal matrix protein import - Suppression of protein import defects in Hansenula polymorpha pex mutants by overproduction of the PTS1 receptor pex5p

NARCIS (Netherlands)

Kiel, JAKW; Veenhuis, M

2000-01-01

In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that

Protein folding includes oligomerization – examples from the endoplasmic reticulum and cytosol

NARCIS (Netherlands)

Christis, C.; Lubsen, N.H.; Braakman, I.

2008-01-01

A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma

Characterization of organic matrix extracted from fresh water pearls

International Nuclear Information System (INIS)

Ma Yufei; Gao Yonghua; Feng Qingling

2011-01-01

Aragonite pearl and vaterite pearl from cultured Hyriopsis cumingii in Zhuji (Zhejiang province, China) were chosen for the study. The matrix proteins were extracted using water and weak acid, and classified as water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM). The proteins from both pearls were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), X-ray diffraction (XRD) and Fourier transformation infrared spectra (FTIR). The results showed that, AIM of aragonite pearl and vaterite pearl had an ordered structure of α-helix. ASM conformations of these two pearls were different from each other. WSM differed the most between these two pearls. - Research Highlights: → We use a specific method for extracting matrix proteins from aragonite pearl and vaterite pearl respectively. → The matrix proteins are extracted by water and weak acid, and classified as water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM). → AIM of aragonite pearl and vaterite pearl have an ordered structure. ASM conformations of the two pearls are different from each other. WSM differ the most between these two pearls.

Changes in the oligomerization potential of the division inhibitor UgtP co-ordinate Bacillus subtilis cell size with nutrient availability.

Science.gov (United States)

Chien, An-Chun; Zareh, Shannon Kian Gharabiklou; Wang, Yan Mei; Levin, Petra Anne

2012-11-01

How cells co-ordinate size with growth and development is a major, unresolved question in cell biology. In previous work we identified the glucosyltransferase UgtP as a division inhibitor responsible for increasing the size of Bacillus subtilis cells under nutrient-rich conditions. In nutrient-rich medium, UgtP is distributed more or less uniformly throughout the cytoplasm and concentrated at the cell poles and/or the cytokinetic ring. Under these conditions, UgtP interacts directly with FtsZ to inhibit division and increase cell size. Conversely, under nutrient-poor conditions, UgtP is sequestered away from FtsZ in punctate foci, and division proceeds unimpeded resulting in a reduction in average cell size. Here we report that nutrient-dependent changes in UgtP's oligomerization potential serve as a molecular rheostat to precisely co-ordinate B. subtilis cell size with nutrient availability. Our data indicate UgtP interacts with itself and the essential cell division protein FtsZ in a high-affinity manner influenced in part by UDP glucose, an intracellular proxy for nutrient availability. These findings support a model in which UDP-glc-dependent changes in UgtP's oligomerization potential shift the equilibrium between UgtP•UgtP and UgtP•FtsZ, fine-tuning the amount of FtsZ available for assembly into the cytokinetic ring and with it cell size. © 2012 Blackwell Publishing Ltd.

Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells

Energy Technology Data Exchange (ETDEWEB)

Celebi, Betuel; Pineault, Nicolas [Hema-Quebec, Research and Development Department, Quebec City, G1V 5C3, PQ (Canada); Mantovani, Diego, E-mail: nicolas.pineault@hema-quebec.qc.ca [Laboratory for Biomaterials and Bioengineering, Department of Materials Engineering and University Hospital Research Center, Laval University, Quebec City, G1V 0A6, PQ (Canada)

2011-10-15

Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells

International Nuclear Information System (INIS)

Celebi, Betuel; Pineault, Nicolas; Mantovani, Diego

2011-01-01

Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

Intergenotypic replacement of lyssavirus matrix proteins demonstrates the role of lyssavirus M proteins in intracellular virus accumulation.

Science.gov (United States)

Finke, Stefan; Granzow, Harald; Hurst, Jose; Pollin, Reiko; Mettenleiter, Thomas C

2010-02-01

Lyssavirus assembly depends on the matrix protein (M). We compared lyssavirus M proteins from different genotypes for their ability to support assembly and egress of genotype 1 rabies virus (RABV). Transcomplementation of M-deficient RABV with M from European bat lyssavirus (EBLV) types 1 and 2 reduced the release of infectious virus. Stable introduction of the heterogenotypic M proteins into RABV led to chimeric viruses with reduced virus release and intracellular accumulation of virus genomes. Although the chimeras indicated genotype-specific evolution of M, rapid selection of a compensatory mutant suggested conserved mechanisms of lyssavirus assembly and the requirement for only few adaptive mutations to fit the heterogenotypic M to a RABV backbone. Whereas the compensatory mutant replicated to similar infectious titers as RABV M-expressing virus, ultrastructural analysis revealed that both nonadapted EBLV M chimeras and the compensatory mutant differed from RABV M expressing viruses in the lack of intracellular viruslike structures that are enveloped and accumulate in cisterna of the degranulated and dilated rough endoplasmic reticulum compartment. Moreover, all viruses were able to bud at the plasma membrane. Since the lack of the intracellular viruslike structures correlated with the type of M protein but not with the efficiency of virus release, we hypothesize that the M proteins of EBLV-1 and RABV differ in their target membranes for virus assembly. Although the biological function of intracellular assembly and accumulation of viruslike structures in the endoplasmic reticulum remain unclear, the observed differences could contribute to diverse host tropism or pathogenicity.

Influence of oligomeric resins on traction and rolling resistance of silica tire treads

NARCIS (Netherlands)

Vleugels, N.; Pille-Wolf, W.; Dierkes, Wilma K.; Noordermeer, Jacobus W.M.

2016-01-01

This study concerns the silica-reinforcement of synthetic rubber compounds for passenger tire treads with the objective to gain insight into the beneficial effects of oligomeric resins, derived from natural and synthetic monomers, on the major tire performance factors: rolling resistance and (wet)

Influence of oligomeric resins on traction and rolling resistance of silica tire treads

NARCIS (Netherlands)

Vleugels, N.; Pille-Wolf, W.; Dierkes, Wilma K.; Noordermeer, Jacobus W.M.

2013-01-01

This study concerns the silica-reinforcement of synthetic rubber compounds for passenger tire treads with the objective to gain insight into the beneficial effects of oligomeric resins, derived from natural and synthetic monomers, on the major tire performance factors: Rolling Resistance and (Wet)

Eicosanoyl-5-hydroxytryptamide (EHT prevents Alzheimer's disease-related cognitive and electrophysiological impairments in mice exposed to elevated concentrations of oligomeric beta-amyloid.

Directory of Open Access Journals (Sweden)

Kesava Asam

Full Text Available Soluble forms of oligomeric beta-amyloid (Aβ are thought to play a central role in Alzheimer's disease (AD. Transgenic manipulation of methylation of the serine/threonine protein phosphatase, PP2A, was recently shown to alter the sensitivity of mice to AD-related impairments resulting from acute exposure to elevated levels of Aβ. In addition, eicosanoyl-5-hydroxytryptamide (EHT, a naturally occurring component from coffee beans that modulates PP2A methylation, was shown to confer therapeutic benefits in rodent models of AD and Parkinson's disease. Here, we tested the hypothesis that EHT protects animals from the pathological effects of exposure to elevated levels of soluble oligomeric Aβ. We treated mice with EHT-containing food at two different doses and assessed the sensitivity of these animals to Aβ-induced behavioral and electrophysiological impairments. We found that EHT administration protected animals from Aβ-induced cognitive impairments in both a radial-arm water maze and contextual fear conditioning task. We also found that both chronic and acute EHT administration prevented Aβ-induced impairments in long-term potentiation. These data add to the accumulating evidence suggesting that interventions with pharmacological agents, such as EHT, that target PP2A activity may be therapeutically beneficial for AD and other neurological conditions.

Hydration effect on the electronic transport properties of oligomeric phenylene ethynylene molecular junctions

International Nuclear Information System (INIS)

Zong-Liang, Li; Huai-Zhi, Li; Yong, Ma; Guang-Ping, Zhang; Chuan-Kui, Wang

2010-01-01

A first-principles computational method based on the hybrid density functional theory is developed to simulate the electronic transport properties of oligomeric phenylene ethynylene molecular junctions with H 2 O molecules accumulated in the vicinity as recently reported by Na et al. [Nanotechnology 18 424001 (2007)]. The numerical results show that the hydrogen bonds between the oxygen atoms of the oligomeric phenylene ethynylene molecule and H 2 O molecules result in the localisation of the molecular orbitals and lead to the lower transition peaks. The H 2 O molecular chains accumulated in the vicinity of the molecular junction can not only change the electronic structure of the molecular junctions, but also open additional electronic transport pathways. The obvious influence of H 2 O molecules on the electronic structure of the molecular junction and its electronic transport properties is thus demonstrated. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

Staphylococcus aureus manganese transport protein C (MntC is an extracellular matrix- and plasminogen-binding protein.

Directory of Open Access Journals (Sweden)

Natália Salazar

Full Text Available Infections caused by Staphylococcus aureus--particularly nosocomial infections--represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM. Manganese transport protein C (MntC, a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA. The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

International Nuclear Information System (INIS)

Wu, Zhengliang L.; Zhou, Hui; Ethen, Cheryl M.; Reinhold, Vernon N.

2016-01-01

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and "3H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain why

Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase

Energy Technology Data Exchange (ETDEWEB)

Wu, Zhengliang L., E-mail: Leon.wu@bio-techne.com [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Zhou, Hui [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States); Ethen, Cheryl M. [Bio-Techne Inc., 614 McKinley Place NE, Minneapolis, MN 55413 (United States); Reinhold, Vernon N., E-mail: Vernon.Reinhold@unh.edu [Gregg Hall, UNH Glycomics Center, University of New Hampshire (United States)

2016-04-29

The 1918 H1N1 influenza virus was responsible for one of the most deadly pandemics in human history. Yet to date, the structure component responsible for its virulence is still a mystery. In order to search for such a component, the neuraminidase (NA) antigen of the virus was expressed, which led to the discovery of an active form (tetramer) and an inactive form (dimer and monomer) of the protein due to different glycosylation. In this report, the N-glycans from both forms were released and characterized by mass spectrometry. It was found that the glycans from the active form had 26% core-6 fucosylated, while the glycans from the inactive form had 82% core-6 fucosylated. Even more surprisingly, the stalk region of the active form was almost completely devoid of core-6-linked fucose. These findings were further supported by the results obtained from in vitro incorporation of azido fucose and {sup 3}H-labeled fucose using core-6 fucosyltransferase, FUT8. In addition, the incorporation of fucose did not change the enzymatic activity of the active form, implying that core-6 fucose is not directly involved in the enzymatic activity. It is postulated that core-6 fucose prohibits the oligomerization and subsequent activation of the enzyme. - Graphical abstract: Proposed mechanism for how core-fucose prohibits the tetramerization of the 1918 pandemic viral neuraminidase. Only the cross section of the stalk region with two N-linked glycans are depicted for clarity. (A) Carbohydrate–carbohydrate interaction on non-fucosylated monomer allows tetramerization. (B) Core-fucosylation disrupts the interaction and prevents the tetramerization. - Highlights: • Expressed 1918 pandemic influenza viral neuraminidase has inactive and active forms. • The inactive form contains high level of core-6 fucose, while the active form lacks such modification. • Core fucose could interfere the oligomerization of the neuraminidase and thus its activation. • This discovery may explain

KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms.

Science.gov (United States)

Aguilar, Claudio; Vlamakis, Hera; Guzman, Alejandra; Losick, Richard; Kolter, Roberto

2010-05-18

Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging and its development for plant protein imaging

Directory of Open Access Journals (Sweden)

Millar A Harvey

2011-07-01

Full Text Available Abstract Matrix-Assisted Laser Desorption/Ionisation (MALDI mass spectrometry imaging (MSI uses the power of high mass resolution time of flight (ToF mass spectrometry coupled to the raster of lasers shots across the cut surface of tissues to provide new insights into the spatial distribution of biomolecules within biological tissues. The history of this technique in animals and plants is considered and the potential for analysis of proteins by this technique in plants is discussed. Protein biomarker identification from MALDI-MSI is a challenge and a number of different approaches to address this bottleneck are discussed. The technical considerations needed for MALDI-MSI are reviewed and these are presented alongside examples from our own work and a protocol for MALDI-MSI of proteins in plant samples.

Mechanics of the mannitol transporter from Escherichia coli : substrate-probing and oligomeric structure

NARCIS (Netherlands)

Veldhuis, Gertjan

2006-01-01

Gertjan Veldhuis bestudeerde de mannitoltransporter van Escherichia coli, EnzymeIImtl (EIImtl), om meer inzicht te krijgen in het bindingsmechanisme en de oligomere toestand van dit membraaneiwit. Hij concludeerde onder andere dat dimeer EIImtl één mannitol bindt, de dimeer zeer stabiel is en dat

Matrix reloaded: CCN, tenascin and SIBLING group of matricellular proteins in orchestrating cancer hallmark capabilities.

Science.gov (United States)

Thakur, Ravi; Mishra, Durga Prasad

2016-12-01

Matricellular proteins (MCPs) are the non-structural extracellular matrix (ECM) proteins with various regulatory functions. MCPs are critical regulators of ECM homeostasis and are often found dysregulated in various malignancies. They interact with various proteins like ECM structural proteins, integrins, growth factor receptors and growth factors to modulate their availability and activity. Cancer-supporting MCPs are known to induce proliferation, migration and invasion of cancer cells. MCPs also support cancer stem (like) cell growth and induce a drug-resistant state. Apart from their direct effects on cancer cells, they play key roles in angiogenesis, immunomodulation, stromal cell infiltration, stromal proliferation and matrix remodeling. High expression of various MCPs belonging to the tenascin, CCN and SIBLING families is often associated with aggressive tumors and poor patient prognosis. Due to their differential expression and distinct functional role, these MCPs are perceived as attractive therapeutic targets in cancer. Studies on preclinical models have indicated that targeting tumor-supportive MCPs could be a potent avenue for developing anti-cancer therapies. The MCP receptors, like integrins and some associated growth factor receptors, are already being targeted using pharmacological inhibitors and neutralizing antibodies. Neutralizing antibodies against CCNs, tenascins and SIBLINGs have shown promising results in preclinical cancer models, suggesting an opportunity to develop anti-MCP therapies to target cancer. Peptides derived from anti-cancer MCPs could also serve as therapeutic entities. In the present review, in continuation with the expanding horizon of MCP functions and disease association, we focus on (i) their unique domain arrangement, (ii) their association with cancer hallmarks and (iii) available and possible therapeutic interventions. Copyright © 2016 Elsevier Inc. All rights reserved.

Polymorphism in SFTPD gene affects assembly and constitutional serum levels of surfactant protein D in a Lebanese population

DEFF Research Database (Denmark)

Fakih, Dalia; Chamat, Soulaima; Medlej-Hashim, Myrna

2014-01-01

Surfactant protein D (SP-D), an oligomeric lung-derived lectin, has essential roles in innate immunity. It can be measured in serum. Previous studies have shown that constitutional SP-D serum levels and the protein degree of multimerization are genetically influenced. We aimed to establish the di...

The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization

Energy Technology Data Exchange (ETDEWEB)

Pan, Yi; Fiscus, Valena; Meng, Wuyi; Zheng, Zhida; Zhang, Lian-Hui; Fuqua, Clay; Chen, Lingling (IMCB-Singapore); (Indiana)

2012-02-08

The Agrobacterium tumefaciens BlcR is a member of the emerging isocitrate lyase transcription regulators that negatively regulates metabolism of {gamma}-butyrolactone, and its repressing function is relieved by succinate semialdehyde (SSA). Our crystal structure showed that BlcR folded into the DNA- and SSA-binding domains and dimerized via the DNA-binding domains. Mutational analysis identified residues, including Phe{sup 147}, that are important for SSA association; BlcR{sup F147A} existed as tetramer. Two BlcR dimers bound to target DNA and in a cooperative manner, and the distance between the two BlcR-binding sequences in DNA was critical for BlcR-DNA association. Tetrameric BlcR{sup F147A} retained DNA binding activity, and importantly, this activity was not affected by the distance separating the BlcR-binding sequences in DNA. SSA did not dissociate tetrameric BlcR{sup F147A} or BlcR{sup F147A}-DNA. As well as in the SSA-binding site, Phe{sup 147} is located in a structurally flexible loop that may be involved in BlcR oligomerization. We propose that SSA regulates BlcR DNA-binding function via oligomerization.

Canine Distemper Virus Matrix Protein Influences Particle Infectivity, Particle Composition, and Envelope Distribution in Polarized Epithelial Cells and Modulates Virulence ▿

OpenAIRE

Dietzel, Erik; Anderson, Danielle E.; Castan, Alexandre; von Messling, Veronika; Maisner, Andrea

2011-01-01

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis o...

Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency.

Science.gov (United States)

Sun, Baoshan; de Sá, Marta; Leandro, Conceição; Caldeira, Ilda; Duarte, Filomena L; Spranger, Isabel

2013-01-30

Recent studies have indicated the presence of significant amount of highly polymerized and soluble proanthocyanidins in red wine and such compounds interacted readily with proteins, suggesting that they might be particularly astringent. Thus, the objective of this work was to verify the astringency of polymeric proanthocyanidins and their contribution to red wine astringency. The precipitation reactions of the purified oligomeric procyanidins (degree of polymerization ranging from 2 to 12-15) and polymeric procyanidins (degree of polymerization ranging from 12-15 to 32-34) with human salivary proteins were studied; salivary proteins composition changes before and after the reaction was verified by SDS-PAGE and procyanidins composition changes by spectrometric, direct HPLC and thiolysis-HPLC methods. The astringency intensity of these two procyanidin fractions was evaluated by a sensory analysis panel. For verifying the correlation between polymeric proanthocyanidins and young red wine astringency, the levels of total oligomeric and total polymeric proanthocyanidins and other phenolic composition in various young red wines were quantified and the astringency intensities of these wines were evaluated by a sensory panel. The results showed that polymeric proanthocyanidins had much higher reactivity toward human salivary proteins and higher astringency intensity than the oligomeric ones. Furthermore, young red wine astringency intensities were highly correlated to levels of polymeric proanthocyanidins, particularly at low concentration range (correlation coefficient r = 0.9840) but not significant correlated to total polyphenols (r = 0.2343) or other individual phenolic compounds (generally r wine astringency and the levels of polymeric polyphenols in red wines may be used as an indicator for its astringency.

Unravelling the nuclear matrix proteome

DEFF Research Database (Denmark)

Albrethsen, Jakob; Knol, Jaco C; Jimenez, Connie R

2009-01-01

The nuclear matrix (NM) model posits the presence of a protein/RNA scaffold that spans the mammalian nucleus. The NM proteins are involved in basic nuclear function and are a promising source of protein biomarkers for cancer. Importantly, the NM proteome is operationally defined as the proteins...

Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Sansom, Mark S P

2015-11-25

The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

Science.gov (United States)

Wellenreuther, G.; Fittschen, U. E. A.; Achard, M. E. S.; Faust, A.; Kreplin, X.; Meyer-Klaucke, W.

2008-12-01

Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

Energy Technology Data Exchange (ETDEWEB)

Wellenreuther, G. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Fittschen, U.E.A. [Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg (Germany); Achard, M.E.S.; Faust, A.; Kreplin, X. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany); Meyer-Klaucke, W. [European Molecular Biology Laboratory, Notkestr. 85, 22603 Hamburg (Germany)], E-mail: Wolfram@embl-hamburg.de

2008-12-15

Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence ({mu}XRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility. A partial separation of protein and internal standard was only observed with untreated silicon, suggesting it may not be an adequate support material. Siliconized quartz proved to be the least prone to heterogeneous drying of the sample and yielded the most reliable results.

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors.

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Manuel Hora

Full Text Available Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS, using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza challenge and controls viral loads better than subunit H5N1 vaccine by eliciting high-affinity antibodies.

Science.gov (United States)

Verma, Swati; Dimitrova, Milena; Munjal, Ashok; Fontana, Juan; Crevar, Corey J; Carter, Donald M; Ross, Ted M; Khurana, Surender; Golding, Hana

2012-11-01

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.

Mueller-matrix mapping of biological tissues in differential diagnosis of optical anisotropy mechanisms of protein networks

Energy Technology Data Exchange (ETDEWEB)

Ushenko, V A; Sidor, M I [Yuriy Fedkovych Chernivtsi National University, Chernivtsi (Ukraine); Marchuk, Yu F; Pashkovskaya, N V; Andreichuk, D R [Bukovinian State Medical University, Chernivtsi (Ukraine)

2015-03-31

We report a model of Mueller-matrix description of optical anisotropy of protein networks in biological tissues with allowance for the linear birefringence and dichroism. The model is used to construct the reconstruction algorithms of coordinate distributions of phase shifts and the linear dichroism coefficient. In the statistical analysis of such distributions, we have found the objective criteria of differentiation between benign and malignant tissues of the female reproductive system. From the standpoint of evidence-based medicine, we have determined the operating characteristics (sensitivity, specificity and accuracy) of the Mueller-matrix reconstruction method of optical anisotropy parameters and demonstrated its effectiveness in the differentiation of benign and malignant tumours. (laser applications and other topics in quantum electronics)

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

Science.gov (United States)

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Pro-inflammatory delipidizing cytokines reduce adiponectin secretion from human adipocytes without affecting adiponectin oligomerization

NARCIS (Netherlands)

Simons, Peter J.; van den Pangaart, Petra S.; Aerts, Johannes M. F. G.; Boon, Louis

2007-01-01

Adiponectin and, especially, its oligomeric complex composition have been suggested to be critical in determining insulin sensitivity. Pro-inflammatory cytokines play an important role in the development of insulin resistance in obesity and associated diseases. Therefore, we investigated the effect

Characterization of a novel Dp71 dystrophin-associated protein complex (DAPC) present in the nucleus of HeLa cells: Members of the nuclear DAPC associate with the nuclear matrix

International Nuclear Information System (INIS)

Fuentes-Mera, Lizeth; Rodriguez-Munoz, Rafael; Gonzalez-Ramirez, Ricardo; Garcia-Sierra, Francisco; Gonzalez, Everardo; Mornet, Dominique; Cisneros, Bulmaro

2006-01-01

Dystrophin is an essential component in the assembly and maintenance of the dystrophin-associated protein complex (DAPC), which includes members of the dystroglycan, syntrophin, sarcoglycan and dystrobrevin protein families. Distinctive complexes have been described in the cell membrane of different tissues and cultured cells. In this work, we report the identification and characterization of a novel DAPC present in the nuclei of HeLa cells, which contains dystrophin Dp71 as a key component. Using confocal microscopy and cell fractionation analyses, we found the presence of Dp71, β-sarcoglycan, β-dystroglycan, α- and β-syntrophin, α1- and β-dystrobrevin and nNOS in the nuclei of HeLa cells. Furthermore, we demonstrated by co-immunoprecipitation experiments that most of these proteins form a complex in the nuclear compartment. Next, we analyze the possible association of the nuclear DAPC with the nuclear matrix. We found the presence of Dp71, β-dystroglycan, nNOS, β-sarcoglycan, α/β syntrophin, α1-dystrobrevin and β-dystrobrevin in the nuclear matrix protein fractions and in situ nuclear matrix preparations from HeLa cells. Moreover, we found that Dp71, β-dystroglycan and β-dystrobrevin co-immunoprecipitated with the nuclear matrix proteins lamin B1 and actin. The association of members of the nuclear DAPC with the nuclear matrix indicates that they may work as scaffolding proteins involved in nuclear architecture

Electrochemistry and biosensing reactivity of heme proteins adsorbed on the structure-tailored mesoporous Nb2O5 matrix

International Nuclear Information System (INIS)

Xu Xin; Tian Bozhi; Zhang Song; Kong Jilie; Zhao Dongyuan; Liu Baohong

2004-01-01

The highly ordered mesoporous niobium oxides fabricated by self-adjusted synthesis have been used as immobilization matrices of heme proteins including Cytochrome c (Cyt C) and horseradish peroxidase (HRP) for their large surface areas, narrow pore size distributions and good biocompatibility. The assembling process was investigated by cyclic voltammetry, amperometry and potential step chronoamperometry in details. Niobium oxide matrices with different structural features were templated with the surfactants and the selectivity of these hosts to specific protein characteristics was determined. It was observed that proteins could be readily assembled onto the mesoporous films with detectable retention of bioactivity. The Nb 2 O 5 matrix with a tailored pore size and counterpoised surface charge to that of hemes allowed for a maximum adsorption capacity of biomolecules. The adsorbed redox molecules exhibited direct electrochemical behavior and gave a pair of well-defined quasi-reversible cyclic voltammetric peaks, indicating that the mesoporous niobium oxide matrix could effectively promote the direct electron transfer between the protein redox site adsorbed and the electrode surface. The midpoint redox potentials of adsorbed Cyt-c and HRP were 14 and -122 mV versus SCE, respectively. Furthermore, the immobilized HRP onto Nb 2 O 5 derived electrode presented good bioactivity and thus was fabricated as an amperometric biosensor for the response of hydrogen peroxide in the range from 0.1 μM to 0.1 mM

Inhibition of haemoglobin-mediated lipid oxidation in washed cod muscle and cod protein isolates by Fucus vesiculosus extract and fraction

DEFF Research Database (Denmark)

Wang, Tao; Jonsdottir, Rosa; Kristinsson, Hordur

2010-01-01

washed cod muscle and protein isolates, phlorotannin-enriched ethyl acetate (EtOAc) fraction showed higher inhibitory effect than crude 80% ethanol (EtOH) extract. The addition of oligomeric phlorotannin-rich subfraction (LH-2) separated by Sephadex LH-20 chromatography, completely inhibited...... similar level of TPC and chemical antioxidant activities as oligomeric subfraction LH-2, it was far less efficient in model systems. These results suggest that other factors rather than the intrinsic reactivity toward radicals could be responsible for the inhibitory effect of phlorotannins on lipid...

Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

Directory of Open Access Journals (Sweden)

Pauline Marie

2015-09-01

Full Text Available Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1 widespread deposition of amorphous calcium carbonate (ACC, (2 ACC transformation into crystalline calcite aggregates, (3 formation of larger calcite crystal units and (4 rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

Science.gov (United States)

Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

2015-09-01

Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

Two overlapping domains of a lyssavirus matrix protein that acts on different cell death pathways.

Science.gov (United States)

Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

2010-10-01

The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control.

Efficacy of enamel matrix protein applied to spontaneous periodontal disease in two dogs.

Science.gov (United States)

Watanabe, Kazuhiro; Kikuchi, Masahiro; Okumura, Masahiro; Kadosawa, Tsuyoshi; Fujinaga, Toru

2003-09-01

Enamel matrix protein (EMP) was applied for regeneration of periodontal tissue in 2 dogs with spontaneous periodontal disease. Case 1 had bony resorption around the root and root apex of the maxillary fourth premolars. Case 2 had vertical resorption of bone between the mandibular first and second molars. A flap was formed in the buccal gingiva, and EMP was applied onto the surface of the exposed root. One or 4 months postoperatively, increased bone level and clinical attachment were recognized. EMP was therefore suggested to be effective to induce regeneration of periodontal tissues in the cases with periodontal disease.

Self-association of the Lentivirus protein, Nef

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Garcia J Victor

2010-09-01

Full Text Available Abstract Background The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization. Results By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123. Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIVMAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface. Conclusions We have

Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

Science.gov (United States)

Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

2013-09-01

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

Integrins and extracellular matrix in mechanotransduction

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Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Crystal structures from the Plasmodium peroxiredoxins: new insights into oligomerization and product binding.

Science.gov (United States)

Qiu, Wei; Dong, Aiping; Pizarro, Juan C; Botchkarsev, Alexei; Min, Jinrong; Wernimont, Amy K; Hills, Tanya; Hui, Raymond; Artz, Jennifer D

2012-03-19

Plasmodium falciparum is the protozoan parasite primarily responsible for more than one million malarial deaths, annually, and is developing resistance to current therapies. Throughout its lifespan, the parasite is subjected to oxidative attack, so Plasmodium antioxidant defences are essential for its survival and are targets for disease control. To further understand the molecular aspects of the Plasmodium redox system, we solved 4 structures of Plasmodium peroxiredoxins (Prx). Our study has confirmed PvTrx-Px1 to be a hydrogen peroxide (H2O2)-sensitive peroxiredoxin. We have identified and characterized the novel toroid octameric oligomer of PyTrx-Px1, which may be attributed to the interplay of several factors including: (1) the orientation of the conserved surface/buried arginine of the NNLA(I/L)GRS-loop; and (2) the C-terminal tail positioning (also associated with the aforementioned conserved loop) which facilitates the intermolecular hydrogen bond between dimers (in an A-C fashion). In addition, a notable feature of the disulfide bonds in some of the Prx crystal structures is discussed. Finally, insight into the latter stages of the peroxiredoxin reaction coordinate is gained. Our structure of PyPrx6 is not only in the sulfinic acid (RSO2H) form, but it is also with glycerol bound in a way (not previously observed) indicative of product binding. The structural characterization of Plasmodium peroxiredoxins provided herein provides insight into their oligomerization and product binding which may facilitate the targeting of these antioxidant defences. Although the structural basis for the octameric oligomerization is further understood, the results yield more questions about the biological implications of the peroxiredoxin oligomerization, as multiple toroid configurations are now known. The crystal structure depicting the product bound active site gives insight into the overoxidation of the active site and allows further characterization of the leaving group

Differential effects of BMP-2 and TGF-beta1 on chondrogenic differentiation of adipose derived stem cells

DEFF Research Database (Denmark)

Mehlhorn, A T; Niemeyer, P; Kaschte, K

2007-01-01

transcriptional regulation of Dlx-5, Msx-2 and Runx-2. MATERIALS AND METHODS: Encapsulated ASC were cultured for 14 days in medium containing TGF-beta1 and/or BMP-2. mRNA expression of the extracellular matrix molecules col2a1, cartilage oligomeric matrix protein, col10a1, alkaline phosphatase (AP......) and transcription factors Msx-2, Dlx-5 and Runx-2 was analysed. Release of glycosaminoglycans, collagen types II and X into the extracellular matrix was demonstrated. RESULTS: BMP-2 and TGF-beta1 induced a chondrogenic phenotype in ASC. Combined growth factor treatment had a synergistic effect on col10a1...

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

Science.gov (United States)

Makarov, M S; Chentsov, Iu S

2010-01-01

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle

Directory of Open Access Journals (Sweden)

Nora López

2012-09-01

Full Text Available The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article.

Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix.

Directory of Open Access Journals (Sweden)

Jessica M Gluck

Full Text Available Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications

Directory of Open Access Journals (Sweden)

Silvia Vilasi

2018-01-01

Full Text Available Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM, is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on

Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth.

Science.gov (United States)

Foley, Joseph; Hill, Shannon E; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin

2013-09-28

Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the

Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth

Science.gov (United States)

Foley, Joseph; Hill, Shannon E.; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin

2013-09-01

Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the

Preparation of mixed matrix adsorber membranes for protein recovery

NARCIS (Netherlands)

Avramescu, M.E.; Girones nogue, Miriam; Borneman, Zandrie; Wessling, Matthias

2003-01-01

This paper presents a generic technology allowing the incorporation of functional entities into a porous substrate. Various ion exchange particles were incorporated into an ethylene vinyl alcohol (EVAL) copolymer porous matrix by an immersion phase separation process and a heterogeneous matrix,

GOLGA2/GM130, cis-Golgi Matrix Protein, is a Novel Target of Anticancer Gene Therapy

OpenAIRE

Chang, Seung-Hee; Hong, Seong-Ho; Jiang, Hu-Lin; Minai-Tehrani, Arash; Yu, Kyeong-Nam; Lee, Jae-Ho; Kim, Ji-Eun; Shin, Ji-Young; Kang, Bitna; Park, Sungjin; Han, Kiwon; Chae, Chanhee; Cho, Myung-Haing

2012-01-01

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an im...

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

Energy Technology Data Exchange (ETDEWEB)

Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

2017-06-12

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

Resonance assignments of the myristoylated Y28F/Y67F mutant of the Mason-Pfizer monkey virus matrix protein

Czech Academy of Sciences Publication Activity Database

Doležal, Michal; Hrabal, R.; Ruml, T.; Rumlová, Michaela

2015-01-01

Roč. 9, č. 2 (2015), s. 229-233 ISSN 1874-2718 Institutional support: RVO:61388963 Keywords : isotopic labeling * matrix protein * M-PMV * myristoylation * resonance assignment * reverse labeling Subject RIV: CE - Biochemistry Impact factor: 0.687, year: 2015

Understanding the Influence of oligomeric resins on traction and rolling resistance of silica tire treads

NARCIS (Netherlands)

Vleugels, N.; Pille-Wolf, W.; Dierkes, Wilma K.; Noordermeer, Jacobus W.M.

2015-01-01

This study concerns the silica reinforcement of styrene–butadiene rubber compounds for passenger car tire treads, with the objective of gaining greater insight into the beneficial effects of oligomeric resins. The major tire performance factors predicted are rolling resistance and (wet) skid

Conformational dynamics of amyloid proteins at the aqueous interface

Science.gov (United States)

Armbruster, Matthew; Horst, Nathan; Aoki, Brendy; Malik, Saad; Soto, Patricia

2013-03-01

Amyloid proteins is a class of proteins that exhibit distinct monomeric and oligomeric conformational states hallmark of deleterious neurological diseases for which there are not yet cures. Our goal is to examine the extent of which the aqueous/membrane interface modulates the folding energy landscape of amyloid proteins. To this end, we probe the dynamic conformational ensemble of amyloids (monomer prion protein and Alzheimer's Ab protofilaments) interacting with model bilayers. We will present the results of our coarse grain molecular modeling study in terms of the existence of preferential binding spots of the amyloid to the bilayer and the response of the bilayer to the interaction with the amyloid. NSF Nebraska EPSCoR First Award

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Science.gov (United States)

Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

2017-08-01

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

Change in the enzymatic dual function of the peroxiredoxin protein by gamma irradiation

International Nuclear Information System (INIS)

An, Byung Chull; Lee, Seung Sik; Lee, Jae Taek; Park, Chul-Hong; Lee, Sang Yeol; Chung, Byung Yeoup

2012-01-01

PP1084 protein was exposed to gamma irradiation ranging from 5 to 500 kGy. Native PAGE showed minor structural changes in PP1084 at 5 kGy, and major structural changes at >15 kGy. Size-exclusion chromatography (SEC) showed the formation of a new shoulder peak when the protein was irradiated with 15 and 30 kGy, and a double peak appeared at 100 kGy. The results of PAGE and SEC imply that PP1084 protein is degraded by gamma irradiation, with simultaneous oligomerization. PP1084 chaperone activity reached the highest level at 30 kGy of gamma irradiation, and then, decreased in a dose-dependent manner with increasing gamma irradiation. However, the peroxidase activity significantly decreased following exposure to all intensities of gamma irradiation. The improvement of chaperone activity using gamma irradiation might be promoted by the oligomeric structures containing covalently cross-linked amino acids. Consequently, PP1084 modification using gamma irradiation could elevate chaperone activity by about 3–4 folds compared to the non-irradiated protein. - Highlights: ► The structure of PP1084 protein was drastically changed above 15 kGy gamma irradiation. ► PP1084 chaperone activity reached the highest level at 30 kGy of gamma irradiation. ► PP1084 modification using gamma irradiation could elevate chaperone activity by about 3–4 folds.

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin

DEFF Research Database (Denmark)

Sasaki, T; Brakebusch, C; Engel, J

1998-01-01

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resis...... in the extracellular matrix of several mouse tissues....... in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1...

Morphology and Phase Transitions in Styrene-Butadiene-Styrene Triblock Copolymer Grafted with Isobutyl Substituted Polyhedral Oligomeric Silsesquioxanes (Postprint)

National Research Council Canada - National Science Library

Drazowski, Daniel B; Lee, Andre; Haddad, Timothy S

2007-01-01

Two symmetric triblock polystyrene-butadiene-polystyrene (SBS) copolymers with different styrene content were grafted with varying amounts of isobutyl-substituted polyhedral oligomeric silsesquioxane (POSS) molecules...

Morphology and Phase Transitions in Styrene-Butadiene-Styrene Triblock Copolymer Grafted with Isobutyl Substituted Polyhedral Oligomeric Silsesquioxanes (preprint)

National Research Council Canada - National Science Library

Drazkowski, Daniel B; Lee, Andre; Haddad, Timothy S

2006-01-01

Two symmetric triblock polystyrene-butadiene-polystyrene (SBS) copolymers with different styrene content were grafted with varying amounts of isobutyl-substituted polyhedral oligomeric silsesquioxane (POSS) molecules...

Catalytic Oligomerization of Terminal Alkynes by Lanthanide Carbyls (η5-C5Me5)2LnCH(SiMe3)2 (Ln = Y, La, Ce)

NARCIS (Netherlands)

Heeres, H.J.; Teuben, J.H.

1991-01-01

Lanthanide and group 3 carbyls Cp*2LnCH(SiMe3)2 (1, Ln = Y; 2, Ln = La; 3, Ln = Ce) are active catalyst precursors for the oligomerization of terminal alkynes HC≡CR (R = alkyl, aryl, SiMe3). The regioselectivity and the extent of oligomerization depend strongly on the lanthanide applied as well as

Two Overlapping Domains of a Lyssavirus Matrix Protein That Acts on Different Cell Death Pathways ▿

Science.gov (United States)

Larrous, Florence; Gholami, Alireza; Mouhamad, Shahul; Estaquier, Jérôme; Bourhy, Hervé

2010-01-01

The lyssavirus matrix (M) protein induces apoptosis. The regions of the M protein that are essential for triggering cell death pathways are not yet clearly defined. We therefore compared the M proteins from two viruses that have contrasting characteristics in terms of cellular apoptosis: a genotype 3 lyssavirus, Mokola virus (MOK), and a genotype 1 rabies virus isolated from a dog from Thailand (THA). We identified a 20-amino-acid fragment (corresponding to positions 67 to 86) that retained the cell death activities of the full-length M protein from MOK via both the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and inhibition of cytochrome c oxidase (CcO) activity. We found that the amino acids at positions 77 and 81 have an essential role in triggering these two cell death pathways. Directed mutagenesis demonstrated that the amino acid at position 77 affects CcO activity, whereas the amino acid at position 81 affects TRAIL-dependent apoptosis. Mutations in the full-length M protein that compromised induction of either of these two pathways resulted in delayed apoptosis compared with the time to apoptosis for the nonmutated control. PMID:20631119

Two-dimensional electrophoretic analysis of nuclear matrix proteins in human colon adenocarcinoma.

Science.gov (United States)

Toumpanaki, A; Baltatzis, G E; Gaitanarou, E; Seretis, E; Toumpanakis, C; Aroni, K; Kittas, Christos; Voloudakis-Baltatzis, I E

2009-01-01

The aim of the present study was to observe possible qualitative and quantitative expression differences between nuclear matrix proteins (NMPs) of human colon adenocarcinoma and their mirror biopsies, using the technique of two-dimensional gel electrophoresis, in order to identify the existence of specific NMP fingerprints for colon cancer. Colon tissues were examined ultrastructurally and NMPs were isolated biochemically, by serial extraction of lipids, soluble proteins, DNA, RNA, and intermediate filaments and were separated according to their isoelectric point (pI) and their molecular weight (MW) by high-resolution two-dimensional electrophoresis (2D). By comparing the 2D electropherograms of colon cancer tissues and mirror biopsy tissues we observed qualitative and quantitative expression differences between their NMPs but also a differentiation of NMP composition between the stages of malignancy. Moreover, despite the similarities between mirror biopsy samples, a highlight percentage of exception was observed. Electrophoretic results provided in this study demonstrated that the examined NMPs could be further investigated as potential markers for detection of colorectal cancer in an early stage, for the assessment of the disease progression, as well as useful tools for individual therapy and for preventing a possible recurrence of cancer and metastasis.

Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

Science.gov (United States)

Dupont-Gillain, C C; Alaerts, J A; Dewez, J L; Rouxhet, P G

2004-01-01

Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.

Science.gov (United States)

Fernandes, Luis G V; Vieira, Monica L; Kirchgatter, Karin; Alves, Ivy J; de Morais, Zenaide M; Vasconcellos, Silvio A; Romero, Eliete C; Nascimento, Ana L T O

2012-10-01

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with K(D) (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a K(D) of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

Science.gov (United States)

Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

2014-01-01

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.