A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis.

During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58-a serine/threonine kinase abundantly expressed during M phase-represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from C. elegans to humans.

Characterization of a Threonine-Rich Cluster in Hepatitis C Virus Nonstructural Protein 5A and Its Contribution to Hyperphosphorylation.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein with key functions in regulating viral RNA replication and assembly. Two phosphoisoforms are discriminated by their different apparent molecular weights: a basally phosphorylated (p56) and a hyperphosphorylated (p58) variant. The precise mechanisms governing p58 synthesis and specific functions of the isoforms are poorly understood. Our study aimed at a deeper understanding of determinants involved in p58 synthesis. We analyzed two variants of p56 and p58 of isolate JFH-1 separately by mass spectrometry using an expression model and thereby identified a threonine-rich phosphopeptide exclusively found in the hyperphosphorylated variant. Individual exchange of possible phosphoacceptor sites to phosphoablatant or -mimetic residues had little impact on HCV replication or assembly in cell culture. A phosphospecific antibody recognizing pT242 revealed that this position was indeed phosphorylated only in p58 and depended on casein kinase Iα. Importantly, phosphoablative mutations at positions T244 and S247 abrogated pT242 detection without substantial effects on global p58 levels, whereas mutations in the preceding serine-rich cluster dramatically reduced total p58 levels but had minor impact on pT242 levels, suggesting the existence of distinct subspecies of hyperphosphorylated NS5A. Mass spectrometry analyses of different genotypes showed variable phosphorylation patterns across NS5A and suggested that the threonine-rich region is also phosphorylated at T242 in gt4a and at S249 in gt1a, gt1b, and gt4a. Our data therefore indicate that p58 is not a single homogenously phosphorylated protein species but rather a population of various phosphoisoforms, with high variability between genotypes.IMPORTANCE Hepatitis C virus infections affect 71 million people worldwide and cause severe chronic liver disease. Recently, efficient antiviral therapies have been established, with inhibitors of nonstructural protein NS5A as a cornerstone. NS5A is a central regulator of HCV replication and assembly but is still enigmatic in its molecular functions. It exists in two phosphoisoforms, p56 and p58. We identified a phosphopeptide exclusively found in p58 and analyzed the determinants involved in phosphorylation of this region. We found evidence for very different phosphorylation patterns resulting in p58. These results challenge the concept of p58 being a homogenous species of NS5A molecules phosphorylated at the same positions and argues for at least two independently phosphorylated variants showing the same electrophoretic mobility, likely serving different functions.

Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis.

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

Insight into the Human DNA Primase Interaction with Template-Primer.

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58C. The specific mode of interaction with a template-primer involving the terminal 5'-triphosphate of RNA and the 3'-overhang of DNA results in a stable complex between p58C and the DNA/RNA duplex. Our results explain how p58C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.

Informative prior distributions for ELISA analyses.

Immunoassays are capable of measuring very small concentrations of substances in solutions and have an immense range of application. Enzyme-linked immunosorbent assay (ELISA) tests in particular can detect the presence of an infection, of drugs, or hormones (as in the home pregnancy test). Inference of an unknown concentration via ELISA usually involves a non-linear heteroscedastic regression and subsequent prediction, which can be carried out in a Bayesian framework. For such a Bayesian inference, we are developing informative prior distributions based on extensive historical ELISA tests as well as theoretical considerations. One consideration regards the quality of the immunoassay leading to two practical requirements for the applicability of the priors. Simulations show that the additional prior information can lead to inferences which are robust to reasonable perturbations of the model and changes in the design of the data. On real data, the applicability is demonstrated across different laboratories, for different analytes and laboratory equipment as well as for previous and current ELISAs with sigmoid regression function. Consistency checks on real data (similar to cross-validation) underpin the adequacy of the suggested priors. Altogether, the new priors may improve concentration estimation for ELISAs that fulfill certain design conditions, by extending the range of the analyses, decreasing the uncertainty, or giving more robust estimates. Future use of these priors is straightforward because explicit, closed-form expressions are provided. This work encourages development and application of informative, yet general, prior distributions for other types of immunoassays.

Skeletal regeneration in the brittle star Amphiura filiformis.

BACKGROUND: Brittle stars regenerate their whole arms post-amputation. Amphiura filiformis can now be used for molecular characterization of arm regeneration due to the availability of transcriptomic data. Previous work showed that specific developmental transcription factors known to take part in echinoderm skeletogenesis are expressed during adult arm regeneration in A. filiformis; however, the process of skeleton formation remained poorly understood. Here, we present the results of an in-depth microscopic analysis of skeletal morphogenesis during regeneration, using calcein staining, EdU labeling and in situ hybridization. RESULTS: To better compare different samples, we propose a staging system for the early A. filiformis arm regeneration stages based on morphological landmarks identifiable in living animals and supported by histological analysis. We show that the calcified spicules forming the endoskeleton first appear very early during regeneration in the dermal layer of regenerates. These spicules then mature into complex skeletal elements of the differentiated arm during late regeneration. The mesenchymal cells in the dermal area express the skeletal marker genes Afi-c-lectin, Afi-p58b and Afi-p19; however, EdU labeling shows that these dermal cells do not proliferate. CONCLUSIONS: A. filiformis arms regenerate through a consistent set of developmental stages using a distalization-intercalation mode, despite variability in regeneration rate. Skeletal elements form in a mesenchymal cell layer that does not proliferate and thus must be supplied from a different source. Our work provides the basis for future cellular and molecular studies of skeleton regeneration in brittle stars.

Neuronal p58IPK Protects Retinal Ganglion Cells Independently of Macrophage/Microglia Activation in Ocular Hypertension.

p58IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma.

Porcine circovirus type 2 exploits cap to inhibit PKR activation through interaction with Hsp40.

Porcine circovirus type 2 is the main pathogen of porcine circovirus disease, which has caused enormous economic losses to the pig industry worldwide. The PKR signaling pathway is important for the cellular antiviral response, but its role in the process of PCV2 infection is unknown. In this study, we first found that dsRNA was produced and that PKR was activated in PCV2 infection. However, interestingly, the activation of PKR was inhibited when the Cap protein was exogenously expressed in PAMs, and this inhibition was reversed by the expression of DNAJC7. The interaction between Cap and DNAJC7 was confirmed by laser confocal microscopy, coimmunoprecipitation and GST pull-down, and it was found that PCV2 infection or the expression of Cap protein could induce DNAJC7 to migrate to the nucleus and release P58IPK, an inhibitor of PKR activation. Downregulating the expression of DNAJC7 by a specific inhibitor or recombinant lentivirus-mediated shRNA, inhibited the replication of the PCV2 genome and the production of virions, which was consistent with the increase of DNAJC7 expression in multiple tissues of weaned piglets infected with PCV2. These data indicate that although PKR was activated by PCV2 infection, the activation was inhibited by Cap through an interaction with DNAJC7. These results help to understand the molecular mechanism of immune escape after PCV2 infection.

p58IPK Is an Endogenous Neuroprotectant for Retinal Ganglion Cells.

p58IPK is an endoplasmic reticulum (ER)-resident chaperone playing a critical role in facilitating protein folding and protein homeostasis. Previously, we have demonstrated that p58IPK is expressed broadly in retinal neurons including retinal ganglion cells (RGCs) and loss of p58IPK results in age-related RGC degeneration. In the present study, we investigate the role of p58IPK in neuroprotection by in vitro and in vivo studies using primary RGC culture and two well-established disease-relevant RGC injury models: retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. Our results demonstrate that in both in vivo models, p58IPK -/- mice exhibit significantly increased RGC loss compared to wild type (WT) mice. In vitro, p58IPK-deficient RGCs show reduced viability and are more susceptible to cell death induced by the ER stress inducer tunicamycin (TM). Overexpression of p58IPK by adeno-associated virus (AAV) significantly diminishes TM-induced cell death in both WT and p58IPK -/- RGCs. Interestingly, we find that loss of p58IPK leads to reduced mRNA expression, but not the protein level, of mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor that resides in the ER. Treatment with recombinant MANF protein protects R28 retinal neural cells and mouse retinal explants from TM-induced cell death. Taken together, our study suggests that p58IPK functions as an endogenous neuroprotectant for RGCs. The mechanisms underlying p58IPK's neuroprotective action and the potential interactions between p58IPK and MANF warrant future investigation.

DNA methylation of the CDC2L1 gene promoter region decreases the expression of the CDK11p58 protein and reduces apoptosis in keloid fibroblasts.

The excessive growth of fibroblasts in keloid is closely related to the status of gene methylation. The aim of this project was to study whether keloid development is related to DNA methylation in the CDC2L1 gene promoter region. DNA methylation of the promoter of this gene was analyzed by bisulfite sequencing and verified by DNA methylation-specific polymerase chain reaction. The results showed that the DNA methylation rate of CpG islands in the CDC2L1 gene promoter region was 50.0% (12/24) in patient keloid tissues and 0% (0/24) in normal skin-tissues from healthy controls. Patient keloid tissues with (n = 12) DNA methylation of the CDC2L1 gene promoter showed higher growth rates than those without (n = 12). Samples from keloid tissues with DNA methylation of the CDC2L1 gene promoter region had dramatically lower levels of CDK11p58 protein than samples from keloid tissues without DNA methylation of the CDC2L1 gene promoter region or healthy normal skin-tissues. In the fibroblasts with DNA methylation of the CDC2L1 gene promoter region from keloid tissues treated with DNA methyl-transferase inhibitor 5 aza 2'-deoxycytidine (5-aza-dC) for 48 h, CDK11p58 levels in the fibroblasts were significantly increased in a dose-dependent manner; the apoptotic rate of the fibroblasts was significantly higher in the treated group than in the non-treated group. This study revealed that DNA methylation exists in the CDC2L1 gene promoter region in keloid tissue fibroblasts. DNA methylation of the CDC2L1 gene promoter region dramatically inhibits the expression of CDK11p58 protein in keloid tissues. A specific demethylation drug, 5-aza-dC, suppressed DNA methylation of the promoter region, which increased the expression of CDK11p58. The elevated expression of CDK11p58 resulted in increased fibroblast apoptosis, thus restraining the development of keloids.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

CDK11p58 Promotes Microglia Activation via Inducing Cyclin D3 Nuclear Localization.

Microglia activation has been implicated in the pathogenesis of many neurological diseases. These reactive microglia are capable of producing a variety of proinflammatory mediators and potentially neurotoxic compounds. The increase of cell number and expression of CD11b are the main features of activated microglia. In this study, we examined the suppressive effects of CDK11p58 on microglia activation induced by lipopolysaccharide (LPS) in vitro. We found that in the activated microglia, the expression of CDK11p58 increased and the overexpression of CDK11p58 could reduce the increased proliferation and CD11b expression in LPS-activated microglia. Such suppressive effects might be resulted from the interaction with cyclin D3 which promoted CDK11p58 nuclear localization. Our results suggested that CDK11p58 acted to regulate microglia activation through CDK11p58 and cyclin D3 interaction.

Overexpression of p58ipk protects neuroblastoma against paraquat-induced toxicity.

BACKGROUND: Paraquat (PQ) is a powerful pathologic pesticide that contribute to the neurotoxicity, however, the pathogenic mechanism between them was unclear. The aims of this study were to explore the underlying mechanism of PQ-induced toxicity and then make potential contribute to such neuronal diseases therapy. METHODS: Human cell line SH-SY5Y was pretreated with a set concentrations of PQ to detect the cell apoptosis and the expression of related genes and proteins. Next, pcDNA 3.1-p58ipk or si-p58ipk was transfected the PQ-induced cells to detect the cytotoxicity. RESULTS: PQ significantly increased the cell apoptosis as well as the expression of p58ipk and CHOP, but decreased the expression of pAKT. p58ipk suppression resulted in an increase of cell apoptosis and CHOP expression, but the expression of pAKT was significantly decreased in PQ-induced SH-SY5Y cells. However, overexpressed p58ipk led to an opposite result. CONCLUSION: The results indicated that the expression of p58ipk was related to the toxicity level of PQ-induced cells and the mechanism between them was that p58ipk regulated the toxicity might through regulating the endoplasmic reticulum stress (ER-stress) and then regulating cell apoptosis. Further studies take emphasize on the effect of ER-stress on neuron system and explore ER-stress-related therapy are important on the treatment of neurodegenerative disease.

GADD45α and γ interaction with CDK11p58 regulates SPDEF protein stability and SPDEF-mediated effects on cancer cell migration.

The epithelium-specific Ets transcription factor, SPDEF, plays a critical role in metastasis of prostate and breast cancer cells. While enhanced SPDEF expression blocks migration and invasion, knockdown of SPDEF expression enhances migration, invasion, and metastasis of cancer cells. SPDEF expression and activation is tightly regulated in cancer cells; however, the precise mechanism of SPDEF regulation has not been explored in detail. In this study we provide evidence that the cell cycle kinase CDK11p58, a protein involved in G2/M transition and degradation of several transcription factors, directly interacts with and phosphorylates SPDEF on serine residues, leading to subsequent ubiquitination and degradation of SPDEF through the proteasome pathway. As a consequence of CDK11p58 mediated degradation of SPDEF, this loss of SPDEF protein results in increased prostate cancer cell migration and invasion. In contrast, knockdown of CDK11p58 protein expression by interfering RNA or SPDEF overexpression inhibit migration and invasion of cancer cells. We demonstrate that CDK11p58 mediated degradation of SPDEF is attenuated by Growth Arrest and DNA damage-inducible 45 (GADD45) α and , two proteins inducing G2/M cell cycle arrest. We show that GADD45 α and γ, directly interact with CDK11p58 and thereby inhibit CDK11p58 activity, and consequentially SPDEF phosphorylation and degradation, ultimately reducing prostate cancer cell migration and invasion. Our findings provide new mechanistic insights into the complex regulation of SPDEF activity linked to cancer metastasis and characterize a previously unidentified SPDEF/CDK11p58/GADD45α/γ pathway that controls SPDEF protein stability and SPDEF-mediated effects on cancer cell migration and invasion.

Identification of p58IPK as a novel neuroprotective factor for retinal neurons.

PURPOSE: Endoplasmic reticulum (ER)-resident chaperone protein p58(IPK) plays a vital role in regulation of protein folding and biosynthesis. The goal of this study was to examine the role of p58(IPK) in retinal neuronal cells under normal and stressed conditions. METHODS: Retinal expression of p58(IPK), retinal morphology, apoptosis, ER stress, and apoptotic gene expression were examined in p58(IPK) knockout (KO) and/or wild-type (WT) mice with or without intravitreal injection of N-methyl-D-aspartic acid (NMDA). In in vitro experiments, differentiated R28 retinal neuronal cells transduced with adenovirus encoding p58(IPK) (Ad-p58(IPK)) or control virus (Ad-LacZ) were exposed to tunicamycin (TM) or hydrogen peroxide (H2O2). Levels of ER stress, apoptosis, and cell survival were evaluated. RESULTS: Chaperone protein p58(IPK) is expressed predominantly in retinal ganglion cells (RGC), inner retinal neurons, and the photoreceptor inner segments. Mice lacking p58(IPK) exhibited increased CHOP expression and loss of RGCs with aging (8-10 months). Intravitreal injection of NMDA induced retinal ER stress and increased p58(IPK) expression in WT mice; this resulted in greater ER stress and enhanced RGC apoptosis in p58(IPK) KO mice. In cultured R28 cells, overexpression of p58(IPK) significantly reduced eIF2α phosphorylation, decreased CHOP expression, and alleviated the activation of caspase-3 and PARP. Overexpression of p58(IPK) also protected against oxidative and ER stress-induced cell apoptosis. Furthermore, p58(IPK) downregulated the proapoptotic gene Bax and upregulated the antiapoptotic gene Bcl-2 expression in stressed R28 cells. CONCLUSIONS: Our study has demonstrated a protective role of p58(IPK) in retinal neurons, which may act in part through a mechanism involving modulation of ER homeostasis and apoptosis, particularly under conditions of cellular stresses.

Hepsin inhibits CDK11p58 IRES activity by suppressing unr expression and eIF-2α phosphorylation in prostate cancer.

Hepsin is a type II transmembrane serine protease frequently overexpressed in prostate cancer (PCa). However, the role of hepsin in PCa remains unclear. In this study, we found that hepsin inhibited the internal ribosome entry site (IRES) activity and expression of CDK11p58, which is associated with cell cycle progression and pro-apoptotic signaling in PCa. Hepsin suppressed CDK11p58 IRES activity in PCa by modulating unr expression and eIF-2α phosphorylation. Further studies revealed that hepsin inhibited the expression of unr by directly binding to unr IRES element and suppressing its activity, and also repressed eIF-2α phosphorylation through down-regulating the expression and phosphorylation of general control non-derepressible-2 (GCN2). Taken together, our data suggest a novel role of hepsin in regulating CDK11p58 IRES activity, and imply that hepsin may act on the machinery of translation to modulate cell cycle progression and survival in PCa cells.

Crystallization and preliminary X-ray diffraction analysis of human DNA primase.

Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space group P1, with unit-cell parameters a = 86.2, b = 88.9, c = 94.68 Å, α = 93.82, ß = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.

Deletion of P58(IPK), the Cellular Inhibitor of the Protein Kinases PKR and PERK, Causes Bone Changes and Joint Degeneration in Mice.

OBJECTIVE: Protein kinase-like endoplasmic reticulum kinase (PERK) and protein kinase R (PKR) are implicated in endoplasmic reticulum stress-induced arthritis and pro-inflammatory cytokine-mediated cartilage degradation in vitro, respectively. We determined whether knockout of the cellular inhibitor of PERK and PKR, P58(IPK) causes joint degeneration in vivo and whether these molecules are activated in human osteoarthritis (OA). MATERIALS AND METHODS: Sections of knee joints from P58(IPK)-null and wild-type mice aged 12-13 and 23-25 months were stained with toluidine blue and scored for degeneration using the osteoarthritis research society international (OARSI) system. Bone changes were assessed by radiology and high-resolution micro-computed tomography of hind limbs. Sections from the medial tibial plateaus of two human knees, removed in total knee replacement surgery for OA, were immunolabelled for phosphorylated PERK and PKR and P58(IPK). RESULTS: Knockout mice exhibited narrower tibiae (p = 0.0031) and smaller epiphyses in tibiae (p = 0.0004) and femora (p = 0.0214). Older knockout mice had reduced total volume inside the femoral periosteal envelope (p = 0.023), reduced tibial (p = 0.03), and femoral (p = 0.0012) bone volumes (BV) and reduced femoral BV fraction (p = 0.025). Compared with wild-types, younger P58(IPK)-null mice had increased OARSI scores in medial femoral condyles (p = 0.035). Thirty four percent of null mice displayed severe joint degeneration with complete articular cartilage loss from the medial compartment and heterotopic chondro-osseous tissue in the medial joint capsule. Phosphorylated PERK and PKR were localized throughout human osteoarthritic tibial plateaus but, in particular, in areas exhibiting the most degeneration. There was limited expression of P58(IPK). CONCLUSION: This study is the first to reveal a critical role for P58(IPK) in maintaining joint integrity in vivo, implicating the PKR and PERK stress signaling pathways in bony changes underlying the pathogenesis of joint degeneration.

Characterization of porcine P58IPK gene and its up-regulation after H1N1 or H3N2 influenza virus infection.

BACKGROUND: The 58-kDa inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (P58IPK) is a cellular protein that is activated during influenza virus infection. Although the function of human P58IPK has been studied for a long time, porcine P58IPK (pP58IPK) has little been studied except for its cloning. OBJECTIVE: In this study, we aimed to investigate the characteristics of the pP58IPK gene, determine its subcellular localization, and find its expression change during H1N1 or H3N2 infection. STUDY DESIGN: First, the sequence and structure of pP58IPK were analyzed. Second, pP58IPK gene was cloned into pEGFP-N1 and pEGFP-C1 vectors, respectively, which were transfected into cells to determine its subcellular localization. Third, Lung tissues of piglets from H1N1 infected, H3N2 infected and control groups were analyzed using histopathology, real-time PCR, and immunohistochemistry. RESULTS: The sequence and structure of pP58IPK was highly similar to the counterpart of human. pP58IPK protein distributed only in the cytoplasm. Lung tissues of piglets infected by H1N1 or H3N2 appeared obvious pathological changes, and the expression of pP58IPK in both mRNA and protein level was up-regulated by approximate 1.5-fold in piglets infected by H1N1 or H3N2 comparing with control piglets. CONCLUSIONS: We analyzed the characteristics of the pP58IPK gene, constructed a phylogenetic tree, determined its subcellular localization, and investigated its expression changes during H1N1 or H3N2 infection. The fundamental data accumulated in this study provides a potential medical model for investigating the function of P58IPK during influenza A viruses infection.

Dynamic expression of P58 IPK in retina of diabetic rats / 中华实验眼科杂志

Background The molecular biological mechanisms of diabetic retinopathy(DR)is unclear up to now.Researches have proved that endoplasmic reticulum stress(ER Stress)-associated factors are elevated in peripheral blood in patients with diabetic retinopathy.and P58 IPK can inhibit those factors.So the relationship between P58 IPK and DR is worth to investigate. Objective The aim of this study was to detect the dynamic expression of P58 IPK in the retina of diabetic rats. Methods The diabetic animal models were established in 18 clean male SD rats by intraperitoneal injection of stilptozotiein(STZ)at a dose of 60 mg/kg.The rats were sacrificed in 1,3,6 months after injection.The expression change of P58 IPK mRNA in the rats retina was detected by quantitative real-time RT-PCR.Other 6 matched normal rats were used as control groups.This experiment followed the Regulations for the Administration of Affair Concerning experimental Animals by State Science and Technology Commission. Results The rats showed more drinking,more food and more urine after STZ injection with the blood glucose level≥ 16.5 mmol/L.The success rate of diabetic models was 100%.The A value of P58 IPK mRNA/β-actin in rat retina was 0.800±0.005 and 0.975±0.008 after injection of STZ.and that of control rats was 0.725±0.006,showing statistically significant difference between them(t=22.589,t=62.784,P＜0.05).In 6 months after injection of STZ,the expression of P58 IPK mRNA in experimental diabetic rat retina was evidently lower than the eontrol rats(0.671±0.004 versus 0.725±0.006,t=-17.984,P＜0.05).Conclusion P58 IPK has a close relation to the pathogenesis of DR,and it plays a retarding role for DR.