乙型肝炎病毒和丙型肝炎病毒蛋白对亮氨酸拉链蛋白LZIP的调节

0 引言乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的感染,不仅引起急、慢性病毒性肝炎,而且与肝纤维化、肝细胞癌(HCC)的发生密切相关.虽然HBV、HCV感染与肝细胞癌之间的关系已经得到确定,但是具体的分子生物学机制还有许多工作要做.其中肝炎病毒编码蛋白对于肝细胞基因组表达的反式调节作用,即肝炎病毒蛋白与肝细胞基因组启动子DNA结合,对于肝细胞基因表达谱产生影响,从而调节肝细胞的生长、代谢、凋亡及恶性转化,在病毒感染致病机制中起着重要作     

丙型肝炎病毒与MAPK信号转导系统

0 引言丙型肝炎病毒(HCV)是一种正链RNA病毒,其致病机制与DNA病毒和逆转录病毒有明显的区别。在病毒的生活周期中,HCV基因组编码多种结构和非结构蛋白,自身结合形成同二聚体形式,或与其他HCV病毒蛋白、细胞蛋白结合形成异二聚体,甚至是多聚体形式在肝细胞内存在。另外,不同的磷酸化形式,还可以发生细胞内转位,对细胞的功能与调节产生影响。在肝细胞质、细胞核中的HCV蛋白对肝细胞正常的信号转导通路进行干扰,这是HCV致病机制的重要组成部分。丝裂原激活蛋白激酶/细胞外信号调节激酶(MAPK/ERK)及其     

乙型和丙型肝炎病毒对MAPKK信号转导的影响

0 引言乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的感染,不仅引起急、慢性病毒性肝炎,而且与肝纤维化、肝细胞癌的发生密切相关。虽然HBV和HCV感染与肝细胞癌之间的关系已经得到确定,但是具体的分子生物     

HBV/HCV重叠感染的抗病毒治疗

从全球范围看,乙型肝炎病毒(hepatitis B virus,HBV)和丙型肝炎病毒(hepatitis C virus,HCV)重叠感染估计约有700-2000万人口感染.重叠感染和单一HBV或HCV感染比较,更易发展为肝硬化、肝细胞癌甚至肝衰竭的比例也高,HBV和HCV重叠感染可有四种不同的临床模式,即HCV活动...     

乙型肝炎病毒和丙型肝炎病毒对纤维连接蛋白基因表达的调节

乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染是病毒性肝炎的主要病原体，感染后引起慢性化的频率较高。在我国，HBV或HCV的感染也是肝硬化和肝细胞癌(HCC)发病的主要原因。纤维连接蛋白(Fibronectin，Fn)是一种存在于血液、体液及各种组织中的具有多种功能的糖蛋白，来源于肝细胞，枯否细胞和内皮细胞，通过与整合素结合调节细胞间的粘附、免疫、凝血和血小板的聚集^[1]，另外在肿瘤与纤维化的发展与发病机制中，Fn也起着重要的作用^[2]。     

乙型肝炎病毒和丙型肝炎病毒对肝细胞信号转导的影响

乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染肝细胞之后,肝炎病毒蛋白的表达对与肝细胞中正常的信号转导通路产生显著的影响.肝炎病毒蛋白对于肝细胞信号转导的影响,势必影响肝细胞的细胞周期、细胞凋亡、细胞生长以及细胞的恶性转化等环节.因为这些细胞的基本的生命活动现象,都与细胞正常的信号转导有关.肝炎病毒蛋白对肝细胞信号调节的影响,一方面通过蛋白一蛋白之间的相互作用而实现,另一方面也通过对肝细胞基因表达谱的影响而实现.因此,利用现代分子生物学技术,对肝炎病毒蛋白结合的蛋白基因进行研究,利用基因差异表达技术对肝炎病毒蛋白反式调节的靶基因进行筛选,阐明肝炎病毒蛋白表达时,能够上调和下调的靶基因类型,对于深入研究肝细胞获得肝炎病毒基因表达之后信号转导途径具有十分重要的意义.中国人民解放军第302医院传染病研究所基因治疗研究中心,在内科传染病学专业博士生导师、生物化学与分子生物学专业博士生导师成军博士、教授、主任医师的领导下,建立了筛选肝炎病毒调节基因结合蛋白的酵母单杂交技术,筛选肝炎病毒蛋白、肝细胞蛋白的结合蛋白的酵母双杂交技术,筛选肝炎病毒蛋白反式调节肝细胞基因的抑制性消减杂交和表达谱基因芯片研究技术等,系统研究肝炎病毒与肝细胞相互作用的分子生物学机制,并有新的发现.本专题研究中所发表的这些重要文章,对于进一步深入探索和研究肝炎病毒对肝细胞正常信号转导的影响,将产生重要推动作用.     

肝炎病毒与肝细胞相互作用的分子生物学机制研究策略

乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染引起的急性和慢性病毒性肝炎、肝硬化、肝细胞癌等,是严重影响中国国民健康的重要传染病。流行病学调查结果表明,在中国HBV表面抗原阳性率为9.75％,HCV抗体的阳性率为3.2％,尽管干扰素、核苷类似物的抗病毒治疗在一部分患者中取得了一定的疗效,中西医结合的综合治疗在终末期肝病的治疗     

乙型肝炎病毒和丙型肝炎病毒相关性肝癌的研究进展

乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)相关的原发性肝细胞癌(HCC)发生机制复杂,其可通过介导机体免疫功能紊乱诱导慢性炎症和产生炎性因子,调控多种癌症信号通路诱导HCC的发生和进展.HBV可通过病毒基因整合、病毒蛋白(主要是HBx、PreS)变异诱导宿主基因不稳定发生插入、缺失等突变诱导HCC的发生和进展.HC...     

丙型肝炎病毒核心蛋白作用研究进展

丙型肝炎病毒(hepatitis C virus,HCV)是一种主要经过血液传播的肝炎病毒,是造成慢性肝炎、肝硬化甚至肝癌的主要原因之一.目前人们对HCV的致病机制的认识仍不很清楚,也缺乏针对HCV有效的治疗方法及疫苗预防.近年研究发现HCV核心(Core,C)蛋白除作为核壳体蛋白具有病毒颗粒组装功能外,还参与调节细胞凋亡、脂代谢、转录以及抗原呈递等作用,与干扰素抵抗也有密切关系.HCV C蛋白具有广泛的反式激活作用,与宿主细胞蛋白相互作用,是导致病毒持续性感染以及肝细胞癌发生的重要原因.深入认知和分析HCV C蛋白分子生物学特性,对阐明HCV持续性感染与致癌机制以及HCV对肝细胞脂肪变与干扰素疗效的影响等诸多问题具有重要意义.     

Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.

Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.     

Characterization of PDZ-binding kinase, a mitotic kinase

hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation.     

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.     

Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain

Most histidine kinases are bifunctional enzymes having both kinase and phosphatase activities. The cytoplasmic kinase domain of EnvZ, a transmembrane histidine kinase functioning as an osmosensor in Escherichia coli, consists of two distinct functional subdomains: domain A [EnvZc(223-289)] and domain B [EnvZc(290-450)]. NMR studies demonstrated that domain A consists of a four-helix bundle serving as a dimerization and phosphotransfer domain, and domain B functions as the ATP-binding and catalytic domain. Here we demonstrate that domain A by itself has the phosphatase activity both in vitro and in vivo. This phosphatase activity is Mg(2+) dependent but is not activated by ADP, ATP, or adenosine 5'-[beta, gamma-imido]triphosphate (AMPPNP), each of which may serve as a cofactor for the EnvZ phosphatase activity. Domain B showed a small but distinct effect on the domain A phosphatase activity only in the presence of ADP or AMPPNP. However, when domain B was covalently linked to domain A, dramatic cofactor-dependent enhancement of the phosphatase activity was observed. Extending domain A for another 75 residues at the C terminus or 44 residues at the N terminus did not enhance its phosphatase activity. Substitution mutations at His-243, the autophosphorylation site, demonstrate that the His residue plays an essential role in the phosphatase activity. The so-called X-region mutant L288P that is known to specifically abolish the phosphatase activity in EnvZ had no effect on the domain A phosphatase function. We propose that the EnvZ phosphatase activity is regulated by relative positioning of domains A and B, which is controlled by external signals. We also propose that the His-243 residue participates in both kinase and phosphatase reactions.     

Mitogen-activated protein kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase

The c-Jun NH₂-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by phosphorylation on Thr and Tyr. Here we report the molecular cloning of a new member of the mammalian MAP kinase kinase group (MKK7) that functions as an activator of JNK. In vitro protein kinase assays demonstrate that MKK7 phosphorylates and activates JNK, but not the p38 or extracellular signal-regulated kinase groups of MAP kinase. Expression of MKK7 in cultured cells causes activation of the JNK signal transduction pathway. MKK7 is therefore established to be a novel component of the JNK signal transduction pathway.     

Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase

Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.     

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.     

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK-substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.