Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, ␣ IIb ␤ 3 , contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteinerich repeats of the ␤ 3 polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC 5.3.4.1) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, ␣ v ␤ 3 , which shares a common ␤-subunit. Thiol isomerase activity within ␣ IIb ␤ 3 is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during ␣ IIb ␤ 3 activation.Integrins are cell-surface, calcium-dependent, heterodimeric adhesion molecules that play a critical role in cell-cell and cell-substrate adhesion. In cells at rest, integrins are present in a latent or resting conformation. Following cellular activation, they undergo conformational changes to become high affinity receptors for their specific ligand(s). The "switch" mechanism whereby integrins are converted from their resting conformation is critically important to their cellular function. However, the mechanisms underlying these conformational changes have not yet been deduced.The conformational changes in the platelet-specific integrin, ␣ IIb ␤ 3 , are the composite result of at least two processes. First, intracellular signals converge on the cytoplasmic tails of the integrin conveying the intention to activate. Second, the extracellular domains, which constitute Ͼ95% of the molecules, respond with an increased affinity for ligand and an altered display of antibody epitopes suggestive of altered protein folding. We have shown that the conserved ␣-subunit cytoplasmic sequence, KVGFFKR, is critical for the intracellular-mediated activation of the platelet integrin (1). The precise role played by this peptide sequence remains uncharacterized. However, Vinogradova et al. (2), have recently proposed a structural basis for this effect which proposes a protein-protein interaction with the integrin cytoplasmic tails. Deletion or mutation of this cytoplasmic sequence from the ␣ IIb subunit were found to increase the ligand binding affinity...

Evidence for involvement of toll-like receptors (TLRs) (e.g. TLR4 and TLR2, whose agonists include lipopolysaccharides (LPS) and saturated fatty acids) in altered patterns of signalling in adipose, liver and muscle from animal models of insulin resistance and obesity has been published. We have now extended this area of research and have determined the effects of LPS on cell viability, insulin secretion, insulin signalling and metabolism in a clonal b-cell line. BRIN-BD11 b-cells were treated for 24 h with increasing concentrations of LPS. Chronic (24 h) and acute (20 min) insulin secretion, insulin content and parameters of cell metabolism and insulin signalling were determined. Incubation of BRIN-BD11 cells for 24 h in the presence of increasing concentrations of the TLR4 ligand LPS significantly decreased chronic (24 h) insulin secretion from 1 . 09G0 . 19 to 0 . 76G0 . 18 mg insulin/mg protein in the presence of 100 ng/ml LPS (P!0 . 05). There was no change in acute (20 min) stimulated insulin secretion or insulin content. Cell metabolism was not changed. Insulin receptor-b (IRb) expression levels were increased significantly from 1G0 . 52 to 8 . 6G1 . 83 units (P!0 . 01), whereas calcineurin activity and Akt phosphorylation were significantly (P!0 . 01 and P!0 . 05 respectively) reduced in response to 24 h incubation in the presence of LPS. There was no change in IR substrate-1 protein expression or phosphorylation after 24 h. Further incubation for 24 h in the absence of LPS resulted in the recovery of chronic insulin secretion. The negative b-cell effects of LPS may contribute to hyperglycaemia in vivo.

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Summary The platelet‐specific integrin αIIbβ3 has endogenous thiol isomerase activity associated with the CXXC motifs within the β subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited αIIbβ3 activation, as detected by PAC‐1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified αIIbβ3 with a 50% inhibitory concentration of 15·5 μmol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin‐independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of αIIbβ3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95 degrees C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.