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Calcium-permeable and thermosensitive transient receptor potential (TRP) channels mediate the nociceptive transduction of noxious temperature in Drosophila nociceptors. However, the underlying molecular mechanisms are not completely understood. Here we find that Subdued, a calcium-activated chloride channel of the Drosophila anoctamin family, functions in conjunction with the thermo-TRPs in thermal nociception. Genetic analysis with deletion and the RNAi-mediated reduction of subdued show that subdued is required for thermal nociception in nociceptors. Further genetic analysis of subdued mutant and thermo-TRP mutants show that they interact functionally in thermal nociception. We find that Subdued expressed in heterologous cells mediates a strong chloride conductance in the presence of both heat and calcium ions. Therefore, our analysis suggests that Subdued channels may amplify the nociceptive neuronal firing that is initiated by thermo-TRP channels in response to thermal stimuli., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca(2+)-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca(2+) that were decondensed upon Ca(2+) depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca(2+) depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca(2+) concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca(2+) and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca(2+) homeostasis.

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

Tripartite motif (TRIM) protein TRIM5alpha has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5alpha is not known. In this study, we showed that transiently overexpressed TRIM5alpha causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5alpha suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5alpha-binding partner. The interaction between Hsp70 and TRIM5alpha was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5alpha-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5alpha, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5alpha-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5alpha. Studies using a series of TRIM5alpha deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5alpha structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5alpha, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5alpha in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5alpha-induced cellular insults.

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO(2)H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO(3)H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H(2)O(2) concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO(3)H) by mass spectrometry. Tsa1p-SO(3)H was not an autoxidation product of Tsa1p-SO(2)H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO(3)H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO(3)H multimer lost its peroxidase activity, it gained approximately 4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO(3)H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO(3)H is a marker of cumulative oxidative stress in cells.

The Ret finger protein (RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger, a B-box, and a coiled-coil domain (together called RBCC). Although RFP is known to become oncogenic when its RBCC moiety is connected to a tyrosine kinase domain by DNA rearrangement, its biological function is not well defined. Here we show that ectopic expression of RFP in human embryonic kidney 293 cells causes extensive apoptosis, as assessed by multiple criteria. RFP expression activates Jun N-terminal kinase and p38 kinase and also increases caspase-3-like activity. However, RFP failed to release cytochrome c and, therefore, to increase caspase-9-like activity. RFP-induced apoptosis could be blocked by the caspase-8 inhibitor crmA and dominant negative ASK1 but not by Bcl-2. These results reveal a novel RFP death pathway that recruits mitogen-activated protein kinase and caspases independently of mitochondrial events. Domain mapping showed that the intact RBCC moiety is necessary for the pro-apoptotic function of RFP. Moreover, expression of the RBCC moiety further potentiated the pro-apoptotic activity and resulted in a 7-fold increase of caspase activation compared with that induced by full-length RFP. This suggests that a large number of tripartite motif family members sharing the RBCC moiety may participate in the control of cell survival.

Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues. Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s). An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay. A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A). The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.

Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H2O2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H2O2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to base line values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by H2O2 might also be required.

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.

A recombinant alpha 1-antitrypsin variant which increased thermal stability was obtained from random mutagenesis followed by screening. The clone was identified as having a single mutation of Phe51-->Cys. Heat deactivation of purified recombinant alpha 1-antitrypsin produced in Escherichia coli revealed that the mutation slowed down the deactivation rate 10-fold at 57 degrees C, increasing thermal stability of recombinant protein to almost that of natural glycosylated plasma form. The mutant protein also exhibited increased stability against denaturant. The urea-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that the mutation shifted midpoint of the transition from 1.9 M to 2.8 M. The mutation site is particularly interesting in that some genetic variants mapped at adjacent positions were shown previously to cause aggregation of the polypeptides, while the Phe51-->Cys mutation decreased aggregation rate significantly during heat deactivation. The association rate constant with porcine pancreatic elastase revealed that the mutation did not affect inhibitory activity significantly. The site identified may be critical for regulating stability of alpha 1-antitrypsin. Characterization of various single amino acid substitutions at position 51 suggests that volume and flexibility of hydrophobic side chain at the site are critical factors for enhancing the stability of alpha 1-antitrypsin.