Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.     

Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.Hepatitis C virus (HCV) is the major causative agent of non-A/non-B hepatitis (26). More than 170 million people, or 3% of the population in the world, are infected with HCV (29). It establishes chronic infection in at least 85% of infected individuals and is associated with liver cirrhosis and hepatocellular carcinoma. Current treatment, which combines polyethylene glycol-interferon (PEG-IFN) and ribavirin, is ineffective in 22% of patients with non-genotype 1 and in 45% of patients with genotype 1 HCV (1, 16, 23, 55). Therefore, identification of new targets for HCV therapy is an important issue, and cellular genes involved in the HCV life cycle may serve as good candidates.HCV is a positive-strand RNA virus and the only known member of Hepacivirus genus in the family Flaviviridae. Its genome has a length of about 9,600 nucleotides coding for a single polyprotein. The long polyprotein is further processed into at least 10 different products, including four structural proteins (core, E1, E2, and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Nonstructural proteins NS3-NS5B are components of the membrane-associated HCV replication complex (8, 13, 36, 45). NS3 is a bifunctional protein containing an N-terminal protease domain and a C-terminal helicase/NTPase domain, and NS4A serves as a cofactor for NS3 protease. NS4B protein is known to induce intracellular membrane changes that probably serve as the site for viral RNA replication (8). NS5A is required for RNA replication, but little is known about its function. NS5B is the RNA-dependent RNA polymerase (reviewed in reference 47).NS5A is phosphorylated on multiple serine and threonine residues and exists in basal phosphorylated (p56) and hyperphosphorylated (p58) forms (49). Increasing evidence suggests that the regulation of NS5A phosphorylation is important for HCV RNA replication. Adaptive mutations or kinase inhibitors, which reduce NS5A hyperphosphorylation, increased the replication of an HCV replicon in cell culture (HCVcc) systems (2, 4, 38). However, when an adaptive replicon with reduced p58 was further treated with the same kinase inhibitor or introduced with a second adaptive mutation, RNA replication was completely blocked (32, 38). Furthermore, the mutations that reduce NS5A hyperphosphorylation and promote RNA replication in cell culture, paradoxically, prevented productive replication in the chimpanzee model (6). These results imply that the tight control of the p58/p56 ratio is important for HCV replication. The detailed mechanism is still not clear, but a clue was provided by the finding of differential association of NS5A phospho-forms with the host vesicle-associated membrane protein-associated protein A (VAP-A) protein, which is an essential molecule for HCV replicase (9, 12). On the other hand, NS5A phosphorylation was recently found to regulate the production of infectious virus (34, 50). Alanine substitutions in the C-terminal domain III of NS5A impaired NS5A phosphorylation, leading to a decrease in NS5A-core protein interaction, disturbance of subcellular localization of NS5A, and disruption of virion production (3, 34, 50). In summary, phosphorylation on NS5A is not only important for HCV RNA replication but also critical for infectious virus production.Since the phosphorylation state of NS5A is correlated with HCV RNA replication and virion production, cellular kinases responsible for NS5A phosphorylation may serve as good candidates for drug targets. Several kinases have been shown to target NS5A in vitro, including casein kinase I (CKI), CKII, MEK1, MKK6, MKK7, AKT, and p70S6K (7, 24). Among these proteins, CKI and CKII are better characterized for NS5A phosphorylation. CKIα has been identified as the target of kinase inhibitors which decrease the hyperphosphorylation of NS5A and was further confirmed as a direct kinase of NS5A (41, 42). CKI requires prephosphorylation of residues near the predicted phosphorylation site in NS5A for effective modification, suggesting that other kinases are also involved in this process (42). CKII has been shown to bind to the C-terminal domain of NS5A and phosphorylate NS5A in vitro (24). Inhibition of CKII with chemical compounds or small interfering RNA (siRNA) did not significantly affect HCV RNA replication but severely disrupted virus production (50).In this study, using lentivirus-based RNA interference (RNAi) screening, we identified a serine/threonine kinase, Polo-like kinase 1 (Plk1), which is involved in HCV replication. Expression of short hairpin RNAs (shRNAs) targeting Plk1 decreased HCV replication and virus production. Moreover, silencing of Plk1 decreased the hyperphosphorylated form of NS5A. In cells treated with a Plk1-specific kinase inhibitor, HCV replication and NS5A hyperphosphorylation were significantly reduced, indicating that Plk1 kinase activity is required for this process. Further studies showed that Plk1 was coimmunoprecipitated and partially colocalized with NS5A, suggesting NS5A as a possible substrate for Plk1. Finally, NS5A is hyperphosphorylated by Plk1 in vitro, supporting the proposition that Plk1 regulates HCV replication through hyperphosphorylation of NS5A.     

Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.     

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4)P recognition site. An extensive interaction study using NMR and other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.     

Complete genome sequence of Mycoplasma pneumoniae type 2a strain 309, isolated in Japan

Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain of this bacterium, has variations in the P1 protein, which is responsible for attachment of the bacterium to host cells. Here, we report the complete genome sequence of M. pneumoniae strain 309 isolated from a pneumonia patient in Japan.     

Proinsulin C-peptide activates α-enolase: implications for C-peptide--cell membrane interaction

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.     

The use of mammalian cultured cells loaded with a fluorescent dye shows specific membrane penetration of undissociated acetic acid

Acetic acid induces unique physiological responses in mammalian cells. Our previous study found that fura-2-loaded human embryonic kidney (HEK) 293T cells showed a robust intracellular fluorescence response immediately after stimulation with acetic acid, and no such response in the case of citric acid. In the present study, we aimed to identify the unique characteristics of acetic acid responsible for this phenomenon. We found that one such feature is its hydrophobicity. We also discovered that acetic acid induces cell responses by intracellular acidification. Of the components of acetic acid in solution (protons, acetate ions, and undissociated acetic acid), undissociated acetic acid might be the functional unit that penetrates the lipid bilayer of cell membranes to acidify the intracellular environment, thereby inducing cell responses. The method used in this study might be convenient in evaluating the intracellular acidification of cultured cells by acids in the external environment.     

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC₅₀ = 7.7–11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.