No abstract

No abstract

Rad23 was identified as a DNA repair protein; although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, ER proteolysis, DNA repair. Rad23 binds the proteasome through a ubiquitin-like (UbL) domain, and contains ubiquitin-associated (UBA) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1), and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated, and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when_UbL is overexpressed, it can compete with Rad23 for proteasome interaction and inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, Threonine-75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.

BackgroundCapsaicin (8-methyl-N-vanillyl-6-nonenamide) is one of the main pungent components of chili peppers and has been shown to exert various effects on numerous physiological processes. Recent studies have focused on the chemopreventive effects of capsaicin, which can combat growth in various human cancer cell systems. The tribbles-related protein 3 (TRIB3) is evolutionarily conserved from Drosophila to humans. In the latter, TRIB3 is a key determinant in numerous cellular processes, including apoptosis.PurposeThe aim of this study was to examine the importance of TRIB3 in the antitumor efficacy of capsaicin in human cancer cells, and further assess potential mechanism(s) underlying the capsaicin-induced upregulation of TRIB3.MethodsHuman cancer cell lines were treated with capsaicin, then evaluated for levels of TRIB3 and molecules related to apoptosis or signaling pathways. The impact of TRIB3 on capsaicin-induced apoptosis was investigated using si-RNA or overexpression of TRIB3.ResultsIt is the first time to show that TRIB3 is targeted by capsaicin to promote apoptosis. Capsaicin promotes apoptotic cell death by upregulating TRIB3 expression in cancer cells. Overexpression of TRIB3 enhances capsaicin-induced apoptosis, and TRIB3 knockdown experiments demonstrate that the effect of capsaicin in apoptotic cell death is correlated with the induction of TRIB3 in cancer cells. Finally, enhancements in gene expression and protein stability are involved in the capsaicin-induced upregulation of TRIB3.ConclusionOur results show that the capsaicin-induced upregulation of TRIB3 triggers apoptosis and thereby contributes to the suppression of cell growth in cancer cell lines.

Abstract. The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC 50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models. IntroductionThe human ribophorin II (RPN2) gene has been localized to chromosome 20ql2-13.1, a region that is frequently deleted in patients with myeloid malignancies (1-4). The gene, which was cloned in 1987 (5), encodes a type I integral membrane protein that is found only in the rough endoplasmic reticulum (ER). Analysis of the structural and topological features of the gene has revealed RPN2 to be a unique integral rough ER membrane glycoprotein that is involved in translocation and the maintenance of the structural uniqueness of the rough ER (5,6). Subsequent biochemical studies have demonstrated that the RPN2 protein is a component of an N-oligosaccharyl transferase complex that conjugates high mannose oligosaccharides to asparagine residues in the N-X-S/T consensus motif of nascent polypeptide chains (7,8).In addition to its association with myeloid disorders, RPN2 has been demonstrated to be a prognostic marker of human breast (9) and pancreatic cancers (10). RPN2 has also been revealed to contribute to the resistance of tumor cells to chemotherapeutic agents, including docetaxel and taxane, in animal models of breast (11) and ovarian (12) ...