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.     

Ipomoelin, a jacalin-related lectin with a compact tetrameric association and versatile carbohydrate binding properties regulated by its N terminus

Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (K(A)) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense.     

Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.     

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif

Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina . The cDNA of P. bellina GDP synthase ( PbGDPS ) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg²⁺ and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.     

Cytoskeleton: actin and endocytosis--no longer the weakest link.

The actin cytoskeleton has long been believed to play a role in endocytosis, but its actual function in this process has been unclear. Now, three proteins that promote actin nucleation have been found to provide a link between the actin cytoskeleton and the endocytic machinery.     

Gamma-tubulin complexes: size does matter.

gamma-Tubulin is a conserved component of all microtubule-organizing centres and is required for these organelles to nucleate microtubule polymerization. However, the mechanism of nucleation is not known. In addition to its localization to organizing centres, a large pool of gamma-tubulin exists in the cytoplasm in a complex with other proteins. The size of the gamma-tubulin complex and number of associated proteins vary among organisms, and the functional significance of these differences is unknown. Recently, the nature of these gamma-tubulin complexes has been explored in different organisms, and this has led us closer to a molecular understanding of microtubule nucleation.     

Escitalopram oxalate induces apoptosis in U‐87MG cells and autophagy in GBM8401 cells

Glioblastoma multiforme (GBM) is recognized as a most aggressive brain cancer with the worst prognosis and survival time. Owing to the anatomic location of gliomas, surgically removing the tumour is very difficult and avoiding damage to vital brain regions during radiotherapy is impossible. Therefore, therapeutic strategies for malignant glioma must urgently be improved. Recent studies have demonstrated that selective serotonin reuptake inhibitors (SSRIs) have cytotoxic effect on certain cancers. Considering as a more superior SSRI, escitalopram oxalate exhibits favourable tolerability and causes generally mild and temporary adverse events. However, limited information is revealed about the influence of escitalopram oxalate on GBM. Therefore, an attempt was made herein to explore the effects of escitalopram oxalate on GBM. The experimental results revealed that escitalopram oxalate significantly inhibits the proliferation and invasive ability of U‐87MG cells and significantly reduced the expressions of cell cycle inhibitors such as Skp2, P57, P21 and P27. Notably, escitalopram oxalate also induced significant apoptotic cascades in U‐87MG cells and autophagy in GBM8401 cells. An animal study indicated that escitalopram oxalate inhibits the proliferation of xenografted glioblastoma in BALB/c nude mice. These findings implied that escitalopram oxalate may have potential in treatment of glioblastomas.     

An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin.

Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+. We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels. Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.     

INFLUENCE OF ULTRAVIOLET RADIATION ON THE EXPRESSION OF PROLIFERATING CELL NUCLEAR ANTIGEN AND DNA POLYMERASE α IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

To evaluate the effects of UV radiation on the expression of DNA replication‐related genes in phytoplankton, the mRNA levels of DNA polymerase α and proliferating cell nuclear antigen (PCNA) in a marine diatom, Skeletonema costatum (Greville) Cleve, were studied using the methods of real‐time quantitative PCR. Treating the algal cultures with UVC radiation for 15 min caused severe mortality during the 24‐h period after treatment. A significant amount of thymine dimers was detected in the treated cultures, and the mRNA levels of DNA polymerase α and PCNA increased by as much as 140 and 23 pmol·(g total RNA)^?1, respectively, compared with the control experiments. In contrast, massive cell deaths did not occur in cultures receiving UVA/B radiation, and the formation of thymine dimers was inconspicuous. Also, UVA/B did not enhance the expression levels of DNA polymerase α or PCNA. Based on the calculation of biologically effective UV doses, daily exposure to sunlight may increase the expression of DNA polymerase α or PCNA genes in S. costatum by 12% at sea surface. This level of increase does not seriously affect the value of using these genes as growth indicators, but caution is needed in the extrapolation of this conclusion to all phytoplankton species.