Mechanism of Integrin Activation by Disulfide Bond Reduction

Yan, Boxu; Smith, Jeffrey W.

doi:10.1021/bi002902i

Cited by 120 publications

(104 citation statements)

References 33 publications

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“…Mild reducing conditions have been shown to be sufficient to bring about integrin activation, the process by which the integrin ligand binding site is uncovered and its affinity increased (66). ␤ 3 integrins, especially ␣ IIb ␤ 3 , were especially sensitive to activation by reducing conditions, since they contain a redox site within the extracellular domain, and sustained activation of ␤ 3 -containing heterodimers required reduction of extracellu-lar free sulfhydryls and disulfide exchange (67,68).…”

Section: Discussionmentioning

confidence: 99%

Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3

Hotchkiss

Ashton

Schwartz

2003

Journal of Biological Chemistry

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Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin ␣ 5 ␤ 1 or ␣ v ␤ 3 , whereas VEGFinduced endothelial cell migration was only blocked by the ␣ v ␤ 3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin ␣ 5 ␤ 1 and, in intact cells, increased the co-localization of focal adhesion kinase with ␣ 5 ␤ 1 . Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to ␣ 5 ␤ 1 and ␣ v ␤ 3 , directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of ␣ 5 ␤ 1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration.

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Section: Discussionmentioning

confidence: 99%

Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3

Hotchkiss

Ashton

Schwartz

2003

Journal of Biological Chemistry

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“…Expression of free sulfhydryls on the extracellular domain of the purified platelet integrin ␣ IIb ␤ 3 in its active conformation was recently demonstrated. 9,18,19 The expression of PDI activity on the surface of cells, including platelets, 20,21 indicates the importance of disulfide exchange on the surface of cells in general and platelets in particular. Furthermore, inhibition of PDI was found to block agonist-induced platelet aggregation.…”

Section: Introductionmentioning

confidence: 99%

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Lahav

Jurk

Hess

et al. 2002

Blood

184

212

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Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor ␣ IIb ␤ 3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified ␣ IIb ␤ 3 , specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in ␣ IIb ␤ 3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to ␣ IIb ␤ 3 as well as ligation-induced allosteric changes in the conformation of ␣ IIb ␤ 3 . We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiolindependent initial ligation followed by a thiol-dependent stabilization of binding. IntroductionThe affinity of integrins for their ligands is tightly regulated, both by cellular events and subsequent to ligand binding. [1][2][3][4][5] Such cellular control over the interaction of extracellular proteins implies the existence of a mechanism to propagate information back and forth between the extracellular head and the cytoplasmic tail of the integrin receptor. In recent years, the posited mechanism has been the allosteric conformational changes occurring in the integrin receptor, both in response to cellular events that switch the receptor from low to high affinity (inside-out signaling) and external events that relay information about occupancy of the receptor (outside-in signaling). [6][7][8][9] Although information about the nature of the changes in conformation is slowly emerging (for review, see Woodside et al 10 ) and consequent changes in intracellular interactions are widely documented, 10-13 the molecular mechanism regulating such changes, particularly on the exofacial domain, has not been elucidated. We previously reported that extracellular sulfhydryls participate in conformational changes triggered by interaction of the platelet integrin ␣ 2 ␤ 1 with its natural ligand collagen. 14 We also found that disulfide exchange is required for platelet adhesion mediated by integrins ␣ 5 ␤ 1 and ␣ IIb ␤ 3 as well as ␣ 2 ␤ 1 and that surface-expressed protein disulfide isomerase (PDI) is involved in this exchange. 15 Furthermore, involvement of disulfide exchange in receptor function is a feature specific to the integrin-receptor family (J. L. et al, submitted manuscript, 2002). We therefore proposed that conformational changes subsequent to ligand interaction with the integrin lead to ...

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“…This reaction has been extensively studied and is known to be important in the function and folding processes of proteins (8)(9)(10). This reaction is also of particular interest because it is known that many proteins that are exposed to mechanical stress in vivo contain disulfide bonds (11)(12)(13). Thus, the effect of force on this reaction could be of significance in biological systems.…”

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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.

322

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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Mechanism of Integrin Activation by Disulfide Bond Reduction

Cited by 120 publications

References 33 publications

Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3

Thymidine Phosphorylase and 2-Deoxyribose Stimulate Human Endothelial Cell Migration by Specific Activation of the Integrins α5β1 and αVβ3

Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

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

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