PYR1/PYL/RCAR proteins (PYLs) are confirmed abscisic acid (ABA) receptors, which inhibit protein phosphatase 2C (PP2C) upon binding to ABA. Arabidopsis thaliana has 14 PYLs, yet their functional distinction remains unclear. Here, we report systematic biochemical characterization of PYLs. A subclass of PYLs, represented by PYL10, inhibited PP2C in the absence of any ligand. Crystal structures of PYL10, both in the free form and in the HAB1 (PP2C)-bound state, revealed the structural basis for its constitutive activity. Structural-guided biochemical analyses revealed that ABA-independent inhibition of PP2C requires the PYLs to exist in a monomeric state. In addition, the residues guarding the entrance to the ligand-binding pocket of these PYLs should be bulky and hydrophobic. Based on these principles, we were able to generate monomeric PYL2 variants that gained constitutive inhibitory effect on PP2Cs. These findings provide an important framework for understanding the complex regulation of ABA signaling by PYL proteins.
Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMNand CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not a-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN-and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-a (TNF-a) and KC co-localized in the liver in the neighbor of HSCs. The double a-SMA-and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for a-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.
De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the ؉1, ؉2, or ؉3 position numbered from the 3 end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the ؉2 and ؉3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the ؉1 position and to maintain the integrity of the 3 end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus. It encodes a single large polyprotein that is subsequently processed into at least 10 separate viral proteins by host as well as virally encoded proteases (17, 21). Of these proteins, nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is responsible for replication of viral RNA genome. Because NS5B is unique to the virus, it is a potential target for developing antiviral drugs and is being subjected to intense research. Recently, several crystal structures of NS5B have been reported, revealing several unique structural features, including a fully encircled active site and a -hairpin structure in the thumb subdomain protruding towards the active site (1, 2, 9). Efforts have been made to characterize NS5B RdRp functions and to understand the mechanistic differences between NS5B and cellular polymerases. This information would help to design direct and specific inhibitors for HCV replication.HCV NS5B is believed to initiate RNA synthesis by a de novo mechanism. De novo initiation of RNA synthesis is common for most v...
VRX0466617 is a novel selective small-molecule inhibitor for Chk2 discovered through a protein kinase screening program. In this study, we provide a detailed biochemical and cellular characterization of VRX0466617. We show that VRX0466617 blocks the enzymatic activity of recombinant Chk2, as well as the ionizing radiation (IR) -induced activation of Chk2 from cells pretreated with the compound, at doses between 5 and 10 Mmol/L. These doses of VRX0466617 inhibited, to some extent, the phosphorylation of Chk2 Ser 19 and Ser 33 -35 , but not of Chk2 Thr 68 , which is phosphorylated by the upstream ataxia-telangiectasia mutated (ATM) kinase. Interestingly, VRX0466617 induced the phosphorylation of Chk2 Thr 68 even in the absence of DNA damage, arising from the block of its enzymatic activity. VRX0466617 prevented the IR-induced Chk2-dependent degradation of Hdmx, concordant with the in vivo inhibition of Chk2. Analysis of ATM/ATM and Rad3-related substrates Smc1, p53, and Chk1 excluded a cross-inhibition of these kinases. VRX0466617 did not modify the cell cycle phase distribution, although it caused an increase in multinucleated cells. Whereas VRX0466617 attenuated IR-induced apoptosis, in short-term assays it did not affect the cytotoxicity by the anticancer drugs doxorubicin, Taxol, and cisplatin. These results underscore the specificity of VRX0466617 for Chk2, both in vitro and in vivo, and support the use of this compound as a biological probe to study the Chk2-dependent pathways. [Mol Cancer Ther 2007;6(3):935 -44]
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