Biochemistry
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Comparative Study
New prospects for an old enzyme: mammalian cytochrome c is tyrosine-phosphorylated in vivo.
Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. ⋯ This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 microM compared to 2.5 microM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.
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The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. ⋯ Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.
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Comparative Study
Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition.
Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. ⋯ With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.
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Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. ⋯ Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.
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A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). ⋯ Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.