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Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis  [PDF]
Wen Wei, Leonie Lampe, Sungha Park, Bhavana S. Vangara, Geoffrey S. Waldo, Stephanie Cabantous, Sarah S. Subaran, Dongmei Yang, Edward G. Lakatta, Li Lin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0050736
Abstract: Background The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor's extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation. Methodology and Result We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation. Conclusion Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface. Significance This is the first report of RAGE intermolecular disulfide bond.
Disulfide Bond Formation and ToxR Activity in Vibrio cholerae  [PDF]
Vera H. I. Fengler, Eva C. Boritsch, Sarah Tutz, Andrea Seper, Hanna Ebner, Sandro Roier, Stefan Schild, Joachim Reidl
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0047756
Abstract: Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.
Structural Insights into the Assembly of CARMA1 and BCL10  [PDF]
Siwei Li, Xue Yang, Juan Shao, Yuequan Shen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042775
Abstract: The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 ?. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.
Prediction Method of Protein Disulfide Bond Based on Pattern Selection  [PDF]
Pengfei Sun, Yuanquan Cui, Tiankai Chen, Ying Zhao
Engineering (ENG) , 2013, DOI: 10.4236/eng.2013.510B083

The effect of the different training samples is different for the classifier when pattern recognition system is established. The training samples were selected randomly in the past protein disulfide bond prediction methods, therefore the prediction accuracy of protein contact was reduced. In order to improve the influence of training samples, a prediction method of protein disulfide bond on the basis of pattern selection and Radical Basis Function neural network has been brought forward in this paper. The attributes related with protein disulfide bond are extracted and coded in the method and pattern selection is used to select training samples from coded samples in order to improve the precision of protein disulfide bond prediction. 200 proteins with disulfide bond structure from the PDB database are encoded according to the encoding approach and are taken as models of training samples. Then samples are taken on the pattern selection based on the nearest neighbor algorithm and corresponding prediction models are set by using RBF neural network. The simulation experiment result indicates that this method of pattern selection can improve the prediction accuracy of protein disulfide bond.

A Prediction Method of Protein Disulfide Bond Based on Hybrid Strategy  [PDF]
Pengfei Sun, Yunhong Ding, Yuyan Huang, Lei Zhang
Journal of Biomedical Science and Engineering (JBiSE) , 2016, DOI: 10.4236/jbise.2016.910B015
A prediction method of protein disulfide bond based on support vector machine and sample selection is proposed in this paper. First, the protein sequences selected are en-coded according to a certain encoding, input data for the prediction model of protein disulfide bond is generated; Then sample selection technique is used to select a portion of input data as training samples of support vector machine; finally the prediction model training samples trained is used to predict protein disulfide bond. The result of simulation experiment shows that the prediction model based on support vector ma-chine and sample selection can increase the prediction accuracy of protein disulfide bond.
Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli
Feras Hatahet, Van Dat Nguyen, Kirsi EH Salo, Lloyd W Ruddock
Microbial Cell Factories , 2010, DOI: 10.1186/1475-2859-9-67
Abstract: Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p.Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.Disulfide bond formation is one of the most common types of protein post-translational modification, with disulfide bonds being found in most outer membrane or secreted proteins. The formation of native disulfide bonds is not trivial and complex pathways have evolved in the three cellular compartments in which catalyzed disulfide bond formation commonly occurs, the endoplasmic reticulum (ER) of eukaryotes [1], the inter-membrane space of mitochondria [2] and the periplasm of prokaryotes [3]. These pathways include components that catalyze the formation of disulfide bonds and others that catalyze the subsequent rearrangement or isomerization of incorrect or non-native disulfide bonds. Despite the presence of these catalyzed pathways for forming protein disulfides native disulfide bond formation is often the rate-limiting step in protein folding in vitro and in vivo.In contrast to the compartments in which catalyzed disulfide bond formation occurs, the environment of the cytoplasm of most prokaryotes has evolved not only lacking components that catalyze formation of
Behavior of a frustrated quantum spin chain with bond dimerization  [PDF]
Tota Nakamura,Satoshi Takada
Physics , 1996, DOI: 10.1103/PhysRevB.55.14413
Abstract: We clarified behavior of the excitation gap in a frustrated S=1/2 quantum spin chain with bond dimerization by using the numerical diagonalization of finite systems and a variational approach. The model interpolates between the independent dimer model and the S=1 spin chain by changing a strength of the dimerization. The energy gap is minimum at the fully-frustrated point, where a localized kink and a freely mobile anti-kink govern the low-lying excitations. Away from the point, a kink and an antikink form a bound state by an effective triangular potential between them. The consequential gap enhancement and the localization length of the bound state is obtained exactly in the continuous limit. The gap enhancement obeys a power law with exponent 2/3. The method and the obtained results are common to other frustrated double spin-chain systems, such as the one-dimensional J_1 - J_2 model, or the frustrated ladder model.
Stabilization of the Single-Chain Fragment Variable by an Interdomain Disulfide Bond and Its Effect on Antibody Affinity  [PDF]
Jian-Xin Zhao,Lian Yang,Zhen-Nan Gu,Hai-Qin Chen,Feng-Wei Tian,Yong-Quan Chen,Hao Zhang,Wei Chen
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12010001
Abstract: The interdomain instability of single-chain fragment variable (scFv) might result in intermolecular aggregation and loss of function. In the present study, we stabilized H4—an anti-aflatoxin B 1 (AFB 1) scFv—with an interdomain disulfide bond and studied the effect of the disulfide bond on antibody affinity. With homology modeling and molecular docking, we designed a scFv containing an interdomain disulfide bond between the residues H44 and L100. The stability of scFv (H4) increased from a GdnHCl 50 of 2.4 M to 4.2 M after addition of the H44-L100 disulfide bond. Size exclusion chromatography revealed that the scFv (H44-L100) mutant existed primarily as a monomer, and no aggregates were detected. An affinity assay indicated that scFv (H4) and the scFv (H44-L100) mutant had similar IC 50 values and affinity to AFB 1. Our results indicate that interdomain disulfide bonds could stabilize scFv without affecting affinity.
Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli
Van Nguyen, Feras Hatahet, Kirsi EH Salo, Eveliina Enlund, Chi Zhang, Lloyd W Ruddock
Microbial Cell Factories , 2011, DOI: 10.1186/1475-2859-10-1
Abstract: Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possibleOur results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.Disulfide bonds are covalent linkages formed between two cysteine residues in proteins. Around one-third of all human proteins fold in the endoplasmic reticulum (ER) and acquire disulfide bonds there. The formation of native disulfide bonds is often the rate limiting step of protein biogenesis [1-3]. Due to this the production of proteins that contain disulfide bonds is difficult, especially on a large scale.In compartments where disulfide bond formation naturally occurs there are two distinct steps in biosynthesis, catalysis of de novo disulfide bond formation and subsequent rearrangement or isomerization to generate native disulfide bonds. In order to produce disulfide bond containing proteins in the cytoplasm of E. coli a variety of strains have been produced [4-11]. These are available commercially under the names origami or rosetta-gami (Novagen) or SHuffle (New England Biolabs) and have the common feacture that they are Δgor ΔtrxB strains and hence have both of the disulfide bond reducing pathways in the cytoplasm disrupted. The SHuffle sys
Preventing Disulfide Bond Formation Weakens Non-Covalent Forces among Lysozyme Aggregates  [PDF]
Vijay Kumar Ravi, Mohit Goel, Hema Chandra Kotamarthi, Sri Rama Koti Ainavarapu, Rajaram Swaminathan
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0087012
Abstract: Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL) at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control) and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ~50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL) on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.
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