Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1

Matthias, Lisa J.; Yam, Patricia T.; Jiang, Xing-Mai; Vandegraaff, Nick; Li, Peng; Poumbourios, Pantelis; Donoghue, Neil; Hogg, Philip J.

doi:10.1038/ni815

Cited by 163 publications

(211 citation statements)

References 47 publications

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“…Indeed, if the surface-associated form of PDI can act as a redox-driven chaperone to unfold proteins after disulfide bond reduction, as has been shown for its endoplasmic reticulum counterpart (38), it may directly catalyze the conformational changes occurring within Env required to ultimately unmask the gp41 fusion peptide. Recently, the reduction of the second domain of CD4 was reported to be an obligatory step in CD4-dependent fusion (39). Our results raise the possibility that redox changes observed within CD4 may be the consequence of thiol/disulfide interchanges occurring within the CD4⅐CXCR4⅐Env complex after Env reduction by PDI.…”

Section: Fig 3 Thiol Content and Cxcr4 Binding Propertiesmentioning

confidence: 52%

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

Barbouche¹,

Miquelis²,

Jones³

et al. 2003

Journal of Biological Chemistry

152

218

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The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

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Section: Fig 3 Thiol Content and Cxcr4 Binding Propertiesmentioning

confidence: 52%

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

Barbouche¹,

Miquelis²,

Jones³

et al. 2003

Journal of Biological Chemistry

152

218

View full text Add to dashboard Cite

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“…It has been shown that thiol͞disulfide exchange in integrin ␣ IIb ␤ III (11) as well as disulfide reduction in von Willebrand factor multimers (37) is necessary for hemostasis and regulating clot formation under high shear forces generated by blood flow. Even the mechanical process of HIV virus fusion and entry into helper T cells has been shown to require disulfide bond reduction in both gp120 of HIV (38) and the CD4 cell surface receptor (39).…”

Section: Resultsmentioning

confidence: 99%

Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques

Wiita

Ainavarapu²,

Huang³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

330

372

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The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol͞disulfide exchange. Reduction of disulfide bonds through the thiol͞disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 Å at the transition state of the thiol͞disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol͞disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.atomic force microscopy ͉ mechanochemistry T he intersection of force and chemistry has been studied for over a century, yet not much is known about this phenomenon compared with more common methods of chemical catalysis (1). There are a number of reasons for this discrepancy, but one of the most important factors remains that it is quite difficult to directly measure the effect of force on a bulk reaction. This difficulty arises because an applied force is not a scalar property of a system; it is associated with a vector. As a result, it is often not possible to directly probe the effect of force on a particular reaction because of heterogeneous application of force and a distribution of reaction orientations (1). To fully quantify the effect of an applied force on a chemical reaction, it is necessary to generate an experimental system where the reaction of interest is consistently oriented with respect to the applied force. Thus, recent advances in single-molecule techniques are particularly well suited to address this problem. The direct manipulation of single molecules allows for the application of force in a vector aligned with the reaction coordinate (2), avoiding the heterogeneity of bulk studies. Earlier works using singlemolecule techniques have described the rupture forces necessary to cleave single covalent bonds (reviewed in ref.

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“…One of several proteins that are susceptible for redox regulation is CD4 itself (11). It might be possible that the CD4-MHC class II interaction is altered upon redox changes in CD4 (28), resulting in different signal transductions into the cell and different outcomes of thymic selection or antigen presentation.…”

Section: Discussionmentioning

confidence: 99%

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

Gelderman

Hultqvist

Holmberg

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

218

199

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Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that the lower capacity to produce ROS is associated with an increased number of reduced thiol groups (؊SH) on T cell membrane surfaces. Artificially increasing the number of reduced thiols on T cells from animals with arthritis-protective Ncf1 alleles by glutathione treatment lowered the threshold for T cell reactivity and enhanced proliferative responses in vitro and in vivo. Importantly, T cells from immunized congenic rats with an E3-derived Ncf1 allele (DA.Ncf1 E3 rats) that cannot transfer arthritis to rats with an arthritis-associated Dark Agouti (DA)-derived mutated Ncf1 allele (DA.Ncf1 DA rats) became arthritogenic after increasing cell surface thiol levels. This finding was confirmed by the reverse experiment, in which oxidized T cells from DA.Ncf1 DA rats induced less severe arthritis compared with controls. Therefore, we conclude that ROS production as controlled by Ncf1 is important in regulating surface redox levels of T cells and thereby suppresses autoreactivity and arthritis development.Ncf1 ͉ reactive oxidative species ͉ p47 phox ͉ NADPH ͉ glutathione R eactive oxygen species (ROS) are generally thought to be harmful and to play a disease-enhancing role in autoimmune diseases such as arthritis (1, 2). However, we have found that a decreased capacity to produce ROS because of polymorphisms in Ncf1 increases susceptibility for autoimmunity and arthritis (3, 4). Ncf1 encodes neutrophil cytosolic factor 1 (Ncf1, also known as p47phox), the activating protein in the NADPH oxidase complex that produces ROS upon activation. In the rat, a SNP in the Ncf1 gene was identified by positional cloning to be one of the strongest genes in regulating both oxidative burst and arthritis (3). In the mouse, a spontaneous mutation was identified that affects splicing and results in expression of truncated, less functional Ncf1 protein (5), which also resulted in increased arthritis and autoimmunity (4). Hence, it was clear that the Ncf1 gene that controls oxidative burst also controlled the autoimmune response and severity of arthritis in both rats and mice.It has been shown that arthritis as induced by immunization with pristane in rats and collagen in mice expressing polymorphic Ncf1 is T cell dependent. In the rat model, only T cells from DA.Ncf1 DA rats [Dark Agouti (DA) rats with the mutated Ncf1 DA allele from the DA rat] can transfer disease to naïve DA.Ncf1 DA recipients, whereas T cells from the congenic DA.Ncf1 E3 rats (DA rats with the WT Ncf1 E3 allele from the E3 rat) cannot (3, 6). In the mouse model, a mutation in Ncf1 results in an increased delayed-type hypersensitivity response and serum levels of anti-collagen type II (CII) IgG antibodies (4), indicating enhanced activation of autoreactive T ...

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Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1

Cited by 163 publications

References 47 publications

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

Protein-disulfide Isomerase-mediated Reduction of Two Disulfide Bonds of HIV Envelope Glycoprotein 120 Occurs Post-CXCR4 Binding and Is Required for Fusion

Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques

T cell surface redox levels determine T cell reactivity and arthritis susceptibility

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