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The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.

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We investigated the role of protein tyrosine phosphatase-alpha (PTPα) expression in the cell death profile of the A431 human carcinoma cell line that was induced by cytotoxic concentrations of the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18). Both NO donors promoted extensive cell detachment in A431 parental cells as compared to the detachment observed for A431 cells that ectopically expressed PTPα (A431 (A27B(PTPα)) cells). The NO-induced cell death characteristics for both cell lines were examined. After incubation for 10 hours with 2.0 mM SNP, attached or detached A431 cells underwent apoptosis. Cells were highly positive for Annexin-V, featured increased cleavage of procaspase-8, activation of downstream caspase-3, and activation of poly-ADP-ribose polymerase 1 (PARP-1). In contrast, exposure of A431 (A27B(PTPα)) cells to 2.0 mM SNP produced an increase in the release of lactate dehydrogenase and enhanced incorporation of propidium iodide. In addition, A431 (A27B(PTPα)) cells showed partial inhibition of the activities of caspase-8, caspase-3, and PARP-1 upon detachment and cell death induced by SNP treatment. Results indicate that necrotic cell damage was induced, characterized by cellular swelling and lysis. We conclude from these results that PTPα regulates the A431 tumor cell death profile mediated by NO donors. Expression of PTPα or its absence may determine the occurrence of NO-induced cell death with necrotic or apoptotic features, respectively.

Hypertension is the most determinant risk factor for cardiovascular diseases. Early intervention and future therapies targeting hypertension mechanisms may improve the quality of life and clinical outcomes. Hypertension has a complex multifactorial aetiology and was recently associated with protein homeostasis (proteostasis). This work aimed to characterize proteostasis in easy-to-access plasma samples from 40 individuals, 20 with controlled hypertension and 20 age- and gender-matched normotensive individuals. Proteostasis was evaluated by quantifying the levels of protein aggregates through different techniques, including fluorescent probes, slot blot immunoassays and Fourier-transform infrared spectroscopy (FTIR). No significant between-group differences were observed in the absolute levels of various protein aggregates (Proteostat or Thioflavin T-stained aggregates; prefibrillar oligomers and fibrils) or total levels of proteostasis-related proteins (Ubiquitin and Clusterin). However, significant positive associations between Endothelin 1 and protein aggregation or proteostasis biomarkers (such as fibrils and ubiquitin) were only observed in the hypertension group. The same is true for the association between the proteins involved in quality control and protein aggregates. These results suggest that proteostasis mechanisms are actively engaged in hypertension as a coping mechanism to counteract its pathological effects in proteome stability, even when individuals are chronically medicated and presenting controlled blood pressure levels.

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