Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity.
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ABSTRACT:
SUBMITTER: Bannikova O
PROVIDER: S-EPMC3561992 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyclophilin J (CyPJ), also called peptidylprolyl isomerase like 3, has been identified as a novel member of the cyclophilin family. Our previous research has resolved the three-dimensional structure of CyPJ and demonstrated the peptidylprolyl cis-trans isomerase (PPIase) activity of CyPJ, which can be inhibited by the common immunosuppressive drug cyclosporine A (CsA). Importantly, CyPJ is upregulated in hepatocellular carcinoma (HCC) and promotes tumor growth; CyPJ inhibition by CsA- or siRNA-based knockdown results in a remarkable suppression of HCC. These findings suggest that CyPJ may be a potential therapeutic target for HCC, and discovery of relevant inhibitors may facilitate development of a novel CyPJ-based targeting therapy. However, apart from the common inhibitor CsA, CyPJ has yet to be investigated as a target for cancer therapy. Here, we report structure-based identification of novel small molecule non-peptidic CyPJ inhibitors and their potential as antitumor lead compounds. Based on computer-aided virtual screening, in silico, and subsequently surface plasmon resonance analysis, 19 potential inhibitors of CyPJ were identified and selected for further evaluation of PPIase CyPJ inhibition in vitro. Thirteen out of 19 compounds exhibited notable inhibition against PPIase activity. Among them, the compound ZX-J-19, with a quinoxaline nucleus, showed potential for tumor inhibition; thus, we selected it for further structure-activity optimization. A total of 22 chemical derivatives with 2,3-substituted quinoxaline-6-amine modifications were designed and successfully synthesized. At least 2 out of the 22 derivatives, such as ZX-J-19j and ZX-J-19l, demonstrated remarkable inhibition of tumor cell growth, comparable to CsA but much stronger than 5-fluorouracil. These results indicate that these two small molecules represent novel potential lead compounds for CyPJ-based antitumor drug development.
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Project description:BACKGROUND: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. RESULTS: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H(2)O(2)) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H(2)O(2). Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. CONCLUSIONS: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro.
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Project description:Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates transcription and coordinates it with pre-mRNA processing. We show here that Rct1, a nuclear multidomain cyclophilin from Schizosaccharomyces pombe, is encoded by an essential gene that interacts with the CTD and regulates its phosphorylation in vivo. Downregulation of Rct1 levels results in increased phosphorylation of the CTD at both Ser2 and Ser5 and in a commensurate decrease in RNAP II transcription. In contrast, overexpression of Rct1 decreases phosphorylation on both sites. The close association of Rct1 with transcriptionally active chromatin suggests a role in regulation of RNAP II transcriptional activity. These data, together with the pleiotropic phenotype upon Rct1 deregulation, suggest that this multidomain cyclophilin is an important player in maintaining the correct phosphorylation code of the CTD and thereby regulating CTD function.
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Project description:We have identified a novel human cyclophilin (hCyP-60) which interacts with the proteinase inhibitor eglin c using the yeast two-hybrid system. A cDNA isolated from a Raji B lymphocyte library reveals a domain showing sequence similarity to known cyclophilins flanked by unique N- and C-terminal residues. In addition, hCyP-60 contains a tyrosine residue (Tyr 389) instead of a tryptophan residue found in most eukaryotic cyclophilins at a position important for cyclosporin binding. Northern and Western analysis reveal widespread expression with considerable tissue-specific variation. Specifically, the highest levels of mRNA are detected in the thymus, pancreas, testis, and K-562 cell line, while the most protein is detected in the kidney. Immunohistochemistry indicates a nuclear-specific localization both in transfected cells and tissue sections. hCyP-60's specific subcellular localization and conserved amino acid sequence suggest that it may play a specific role in the nucleus.
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