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REGULATION OF BPV E1 PROTEIN ACTIVITY BY PHOSPHORYLATION

通过磷酸化调节 BPV E1 蛋白活性

基本信息

批准号：
2594516
负责人：
MICHAEL R LENTZ
金额：
$ 8.84万
依托单位：
UNIVERSITY OF NORTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-04-01 至 2000-08-30
项目状态：
已结题

项目摘要

Papillomaviruses are the causative agents of cutaneous and genital warts in the human population. they are also the likely cause of the majority of cases of human cervical cancer. Viral DNA in cells transformed by these viruses replicates as a stable episome in the nucleus. Papillomavirus DNA replication is tightly regulated, and takes place in synchrony with the host cell DNA. While we now know in some detail the steps leading to viral replication initiation, very little is understood about how papillomavirus DNA replication is regulated. The best characterized papillomavirus system is bovine papillomavirus type-1 (BPV- 1 or BPV). Two viral proteins, E1 and E2, are necessary and sufficient for BPV viral DNA replication. these proteins are likely to function by initiating the assembly of a functional replication complex at the viral replication origin, and by participating in the initial unwinding of the viral DNA. All other enzymatic activities of the replication process are carried out by host cell proteins. E1 is a phosphoprotein that recognizes and binds specifically to the BPV replication origin. In addition to its DNA binding activity, E1 forms a tight complex with the E2 protein, and has both ATPase and helicase activity. The role of the E1/E2 complex in DNA replication is not clear. E2 may alter the conformation of E1 to stimulate specific DNA binding activity. I propose to examine the role phosphorylation of the E1 protein plays in regulating E1 protein and DNA replication activity. We will answer the following questions: 1.) Which amino acids on the E1 protein are phosphorylated? To answer this question, we will use proteolytic digestion and peptide analysis of in vivo labeled E1 protein. 2.) What is the function of specific phosphoamino acids for E1 protein activity? Mutations will be made at phosphorylation sites and the effect of these mutations on the DNA binding, E2 protein binding, enzyme activity and replication activity of the E1 proteins will be determined. 3.) Are there changes in the pattern of E1 phosphorylation in different stages of the cell cycle? Our approach will be to isolate E1 protein from cells in specific stages of the cell cycle and analyze the phosphorylation pattern of these proteins. 4.) Which host cell enzymes control the phosphorylation state of the E1 protein? Antibodies and inhibitors to specific kinases and phosphatases will be used to identify enzymes active on the E1 protein. Host cell activities are known to be required for the control of BPV DNA replication. From our results, we therefore expect to learn something about the regulation of cellular, as well as viral, DNA replication, and mechanisms by which this control may be lost in cancer cells. These results may ultimately lead to new ideas for ways to inhibit the growth of papillomaviruses.

乳头瘤病毒是皮肤疣和生殖器疣的病原体在人口中。它们也是大多数人的可能原因人类宫颈癌病例。转化细胞中的病毒DNA 这些病毒在细胞核中作为稳定的附加体进行复制。乳头瘤病毒 DNA 复制受到严格调控，并发生在与宿主细胞 DNA 同步。虽然我们现在已经知道了一些细节导致病毒复制启动的步骤，人们知之甚少关于乳头瘤病毒 DNA 复制如何受到调节。最好的特征性乳头瘤病毒系统是牛乳头瘤病毒1型（BPV- 1 或 BPV）。两种病毒蛋白 E1 和 E2 是必要且充分的用于 BPV 病毒 DNA 复制。这些蛋白质可能通过启动病毒复制复合体的组装复制起点，并通过参与初始解旋病毒DNA。复制过程的所有其他酶活性都是由宿主细胞蛋白进行。 E1是一种磷蛋白识别并特异性结合 BPV 复制起点。在除了其 DNA 结合活性外，E1 还与 E2蛋白，具有ATP酶和解旋酶活性。的作用 E1/E2复合物在DNA复制中的作用尚不清楚。 E2 可能会改变 E1 构象可刺激特定的 DNA 结合活性。我建议检查 E1 蛋白磷酸化在调节中的作用 E1蛋白和DNA复制活性。我们将回答以下问题问题： 1.) E1 蛋白上的哪些氨基酸被磷酸化？为了回答这个问题，我们将使用蛋白水解消化和肽体内标记E1蛋白的分析。 2.) 的功能是什么 E1 蛋白活性的特定磷酸氨基酸？突变将是在磷酸化位点进行的突变以及这些突变对 DNA 结合、E2 蛋白结合、酶活性和复制活性 E1 蛋白的数量将被确定。 3.) 是否有变化 E1磷酸化在细胞周期不同阶段的模式？我们的方法是从特定阶段的细胞中分离E1蛋白细胞周期并分析这些蛋白质的磷酸化模式。 4.) 哪些宿主细胞酶控制 E1 的磷酸化状态蛋白质？特定激酶和磷酸酶的抗体和抑制剂将用于鉴定对 E1 蛋白有活性的酶。宿主细胞已知控制 BPV DNA 需要活动复制。因此，我们希望从我们的结果中学到一些东西关于细胞以及病毒、DNA 复制的调节，以及癌细胞中可能失去这种控制的机制。这些结果可能最终会带来抑制生长的新想法乳头瘤病毒。