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.
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.
Protein degradation that occurs in tissue during post-mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap-freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2-DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high-molecular-weight proteins and reduction in protein fragmentation.
The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), β-Lg A and β-Lg B were observed as hydrolysis proceeded to a 5 % degree of hydrolysis (DH) in both unheated and heat-treated (80 ºC, 10 min) WPI dispersions (100 g L -1 ). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC-MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate -the majority of which were derived from β-Lg. The mapping of the detected regions in α-La, β-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.
Evidence exists to support the role of dairy derived proteins whey and casein in glycemic management. The objective of the present study was to use a cell screening method to identify a suitable casein hydrolysate and to examine its ability to impact glycemia related parameters in an animal model and in humans. Following screening for the ability to stimulate insulin secretion in pancreatic beta cells, a casein hydrolysate was selected and further studied in the ob/ob mouse model. An acute postprandial study was performed in 62 overweight and obese adults. Acute and long-term supplementation with the casein hydrolysate in in vivo studies in mice revealed a glucose lowering effect and a lipid reducing effect of the hydrolysate (43% reduction in overall liver fat). The postprandial human study revealed a significant increase in insulin secretion ( p = 0.04) concomitant with a reduction in glucose ( p = 0.03). The area under the curve for the change in glucose decreased from 181.84 ± 14.6 to 153.87 ± 13.02 ( p = 0.009). Overall, the data supports further work on the hydrolysate to develop into a functional food product.
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