Biomedical subjects
Publications and source records attributed to Sripadi Prabhakar.
The DNA triplet GCA is successfully used for the first time as a chiral selector for the chiral discrimination and optical purity measurement of some alpha-amino acids by investigating the collision-induced dissociation spectra of the sodiated ternary complex ion formed by electrospray ionization.
Understanding the mechanisms of evolution requires identification of the molecular basis of the multiple (pleiotropic) effects of specific adaptive mutations. We have characterized the pleiotropic effects on protein levels of an adaptive single-base pair substitution in the coding sequence of a signaling pathway gene in the bacterium Pseudomonas fluorescens SBW25. We find 52 proteomic changes, corresponding to 46 identified proteins. None of these proteins is required for the adaptive phenotype. Instead, many are found within specific metabolic pathways associated with fitness-reducing (that is, antagonistic) effects of the mutation. The affected proteins fall within a single coregulatory network. The mutation 'rewires' this network by drawing particular proteins into tighter coregulating relationships. Although these changes are specific to the mutation studied, the quantitatively altered proteins are also affected in a coordinated way in other examples of evolution to the same niche.
In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.
Lentiviral Nef increases T cell signaling activity, but the molecular nature of the stimulus involved is incompletely described. We explored CD4 T cell lipid raft composition in the presence and absence of Nef. Here, the E2 ubiquitin-conjugating enzyme UbcH7, which acts in conjunction with c-Cbl, is absent from lipid rafts. This Nef-mediated exclusion is associated with failure of ubiquitination of activated Vav. In the presence of Nef, lipid raft Cdc42 is activated and forms a ternary complex between the c-Cbl-interacting protein p85Cool-1/betaPix and c-Cbl, displacing UbcH7 from rafts. Suppression of p85Cool-1/betaPix expression restores UbcH7 raft localization and Vav ubiquitination and diminishes Cdc42 activity. Moreover, p85Cool-1/betaPix knockdown attenuates HIV replication. Thresholds for activation of signaling involve the intricate balance of positive and negative regulators. Here we provide evidence for Nef disruption of a negative regulator of T cell signaling in promoting HIV replication.
Uveal malignant melanoma is the most frequent primary intraocular tumor in adult humans. The cellular events leading to neoplasic transformation of normal uveal melanocytes are not well known when compared to other cancers. In this study, we investigated the role of G1 and G1/S regulatory proteins of the cell cycle in human uveal melanoma (UM) primary cell cultures, since these proteins are common targets in tumor development. Further, freshly established and characterized tumor cells are a better model for in vitro studies when compared to cell lines established long ago. Human primary cell cultures from eight different UM were established, as well as one primary culture from rhesus uveal normal melanocytes (UNM). Primary human UM cultures were characterized by a low establishment and growing rate. From four successful cultures, three showed a high expression of cyclin D1, cyclin E, p16NK4A, and p27KIP1 with no variations in cyclin A, cyclin-dependent kinase 2 (CDK2), and CDK4. Interestingly, in one of the cultured tumors, tumor suppressor protein retinoblastoma (Rb) did not bind E2F despite the fact that Rb was found in its hypophosphorylated form. No mutations in either RB1 or the Rb-binding pocket of E2F-1 were detected. Furthermore, we identified seven proteins co-immunoprecipitating with Rb in this tumor, including Lamin A/C and six proteins not previously reported to bind Rb: Hsc70, high mobility group protein 1 (HMG-1), hnRPN, glyceraldehyde 3 phosphate dehydrogenase (G3PDH), EF-1, and EF-2. Our results indicate that the overexpression of cyclins D1/E and CDKIs p16 and p27, together with a deregulation of the Rb/E2F pathway, may be implicated in the development of human UM.
Platelets play a key role in the control of bleeding and wound healing, contributing to the formation of vascular plugs. Under pathologic circumstances, they are involved in thrombotic disorders, including heart disease. Since platelets do not have a nucleus, proteomics offers a powerful alternative approach to provide data on protein expression in these cells, helping to address their biology. In this publication we extend the previously reported analysis of the pI 4-5 region of the human platelet proteome to the pI 5-11 region. By using narrow pI range two-dimensional electrophoresis (2-DE) for protein separation followed by high-throughput tandem mass spectrometry (MS/MS) for protein identification, we were able to identify 760 protein features, corresponding to 311 different genes, resulting in the annotation of 54% of the pI 5-11 range 2-DE proteome map. We evaluated the physicochemical properties and functions of the identified platelet proteome. Importantly, the main group of proteins identified is involved in intracellular signalling and regulation of the cytoskeleton. In addition, 11 hypothetical proteins are reported. In conclusion, this study provides a unique inventory of the platelet proteome, contributing to our understanding of platelet function and building the basis for the identification of new drug targets.
We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2). Further studies revealed that the change in mobility of Dok-2 was brought about by tyrosine phosphorylation. Dok-2 tyrosine phosphorylation was also found to be involved in collagen receptor, glycoprotein VI (GPVI), signaling as well as in outside-in signaling through the major platelet integrin, alpha IIIb beta 3. These studies also provided the first demonstration of posttranslational modification of 2 regulator of G protein signaling (RGS) proteins, RGS10 and 18. Phosphorylation of RGS18 was mapped to Ser49 by MS/MS analysis. This study provides a new approach for the identification of novel signaling molecules in activated platelets, providing new insights into the mechanisms of platelet activation and building the basis for the development of therapeutic agents for thrombotic diseases.