Formation Of Disulfide-linked Dimers Research Articles

The Molecular Basis for Erns Dimerization in Classical Swine Fever Virus.

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein Erns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant Erns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form Erns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of Erns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

Bridging of membrane surfaces by annexin A2

The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca2+ regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca2+ dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.

ERAP1 deficiency mitigates HLA-B27 misfolding and ER stress, and protects HLA-B27 transgenic rats from arthritis and orchitis

Abstract Endoplasmic reticulum aminopeptidase 1 (ERAP1) is associated with ankylosing spondylitis (AS) in HLA-B27-positive individuals. ERAP1 optimizes peptide length for binding to MHC class I molecules. However, the role of HLA-B27-bound peptides in AS pathogenesis remains unclear. We investigated epistasis between ERAP1 and HLA-B27 in a rat model of spondyloarthritis (SpA). ERAP1-deficient rats generated by TALEN-mediated genome editing were crossed with disease prone HLA-B27 transgenic (B27 Tg) rats (33-3 transgene). ERAP1 deficiency reduced the prevalence of arthritis (4/34 in ERAP1−/− vs. 12/37 in ERAP1+/+; p<0.05) and testicular inflammation (6/34 vs. 15/37, p<0.05) in male B27 Tg rats. Wild type and HLA-B7 transgenic (B7 Tg) rats with and without ERAP1 remained healthy. Bone marrow-derived macrophages (BMDMs) from B27 Tg ERAP1−/− rats exhibited a 12-fold (p<0.05) increase in MARB4-reactive cell surface B27 complexes containing long peptides, a 30% reduction of total cellular unfolded B27 heavy chains (HC) (p<0.01), and a 32% increase in the ratio of folded/unfolded B27 HC (p=0.01). ERAP1 deficiency also reduced by 50% the formation of disulfide-linked dimers and B27-BiP complexes (p<0.01). ERAP1-deficiency attenuated BiP and CHOP induction and XBP1 splicing (50%, 27%, and 25%, respectively; p<0.01) caused by IFNg-induced B27 upregulation. These results indicate that ERAP1-deficiency is protective for arthritis development in 33-3 B27 Tg rats, and raise the possibility that alleviation of ER stress may contribute to protection, although further investigation is needed to define the mechanism.

231 - Glutathionylation at the Active Sites of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H2O2 results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin–thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin–thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260–460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.

Structural and functional analysis of native peroxiredoxin 2 in human red blood cells

Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H2O2 results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin–thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin–thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160kDa) oligomers and that decamers or higher order molecular weight oligomers (260–460kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities.

Stress, DNA Damage, and ATM

The protein kinase ATM (ataxia-telangiectasia mutated) is a key component of the signaling pathway through which cells are protected from DNA damage. ATM becomes activated within a protein complex at sites of double-stranded breaks in DNA. ATM is also activated in response to increased production of reactive oxygen species (ROS). Such activation was thought to reflect DNA damage caused by ROS, but Guo et al. (p. [517][1]) showed that ATM was in fact directly activated by ROS. A cysteine residue in ATM contributes to the formation of disulfide-linked dimers of activated ATM on exposure to ROS in vitro. Experiments using mutated forms of the enzyme suggested that two distinct mechanisms regulated ATM activity. [1]: /lookup/doi/10.1126/science.1192912

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

Mutations in the shutter region of anti-thrombin result in severe episodic thrombosis and unlike other serpins in formation of disulfide-linked dimers

Mutations in the shutter region of anti-thrombin result in severe episodic thrombosis and unlike other serpins in formation of disulfide-linked dimers

Constitutive Activation and Specific Signaling of the Xmrk Receptor in Xiphophorus melanoma

Receptor tyrosine kinases (RTKs) have been found to play a role in the initiation and progression of different diseases in mammals. Melanoma formation in the fish Xiphophorus is a well-established genetic model in which an RTK is involved in tumorigenesis. The overexpression of the epidermal growth factor receptor–related RTK Xmrk (Xiphophorus melanoma receptor kinase) is the primary step in this hereditary oncogenic condition. In malignant tumors the Xmrk receptor is constitutively activated due to the presence of high levels of receptor molecules containing two activating mutations. These mutations have been localized in the extracellular domain of the receptor, and both lead to the activation of Xmrk through the formation of disulfide-linked dimers. The identification of different intracellular substrates that interact with the activated receptor revealed that proliferative as well as antiapoptotic pathways are involved in the transformation of the pigment cells.

Dimerization between the holin and holin inhibitor of phage lambda.

Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of lambda-infected cell lysis depends solely on the 107 codon S gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.

Role of Cotranslational Disulfide Bond Formation in the Folding of the Hemagglutinin-Neuraminidase Protein of Newcastle Disease Virus

The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakmanet al.(EMBO J.11, 1717–1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, thet1/2for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.

The Role of Individual Oligosaccharide Chains in the Activities of the HN Glycoprotein of Newcastle Disease Virus

To explore the role of N-linked carbohydrate in the activities of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), the six glycosylation addition sites (G1-G6) in the HN sequence of the AV strain of NDV were mutated. Migration of mutant protein on polyacrylamide gels as well as endoglycosidase H digestion of mutant protein showed that four of the addition sites (G1, G2, G3, and G4 at amino acids 119, 341, 433, and 481, respectively) are used while two (G5 and G6 at amino acids 508 and 538, respectively) are not used. Proteins expressed from single and all possible combinations of double and triple mutant DNAs as well as the unglycosylated molecule were characterized for the presence of specific antigenic sites, formation of disulfide-linked dimers, stability, transport to the cell surface, and biological activity. Results showed that glycosylation at positions G1 and G2 play little detectable role in the folding, stability, or transport of the molecule either singly or in combination with other mutations. Mutation of these sequons, however, significantly increased the cell attachment and fusion promotion activities of the protein, particularly in combination. Mutation of the glycosylation site at G4 either singly or in combination with other site-eliminating mutants inhibited the formation of the mature protein, while a mutation eliminating the addition site at G3 had a slight effect on the efficiency of folding, particularly in combination with mutation of the site G4. When normalized to surface expression, elimination of carbohydrate addition sites at G3 and G4 singly or in combination with other mutations depressed in particular the neuraminidase activity of the protein but not the fusion promotion activity. Thus two oligosaccharides do not have a detectable role in maturation but do modulate the biological activities of the protein. The other two oligosaccharides influence both folding and activity of the protein.