BLEOMYCIN HYDROLASES--PROTEASES THAT BIND DNA

博莱霉素水解酶——结合 DNA 的蛋白酶

• 批准号: 6146622
• 负责人: Leemor Joshua-Tor
• 金额: $ 23.97万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6146622
• 关键词: DNA; DNA binding protein; X ray crystallography; active sites; bleomycin; crystallization; enzyme activity; enzyme substrate complex; gel electrophoresis; hydrolysis; oligonucleotides; peptidases; peptide chemical synthesis

Resistance to anti-neoplastic drugs is a central problem in treatment of human cancers. The cysteine protease bleomycin hydrolase was first recognized due to its ability to hydrolyze the anti-cancer drug bleomycin. The recent discovery of homologs of this protease in yeast and bacteria suggest a conserved cellular function for this protein, yet, the only known natural substrate, bleomycin, is almost never seen by any of these cells. The yeast homolog, Gal6 is an intracellular, primarily nuclear, protease that binds DNA and acts as a repressor in the Gal4 regulatory system. The cellular function of this ubiquitously-expressed protein is still unknown. In recent years, there has been considerable interest in intracellular proteases, since they seem to be involved in important regulatory processes. Bleomycin hydrolase, with its many functions including its unusual DNA-binding activity adds another dimension of complexity of these proteases. The crystal structure of the yeast bleomycin hydrolase, Gal6, revealed a hexameric structure in which the protease and DNA- binding activities are structurally intertwined. The goal of this project is to understand the functional coupling of these two activities in GaI6. These studies will foster an understanding of how bleomycin hydrolase functions in tumor drug resistance and may provide insight to what are its basic cellular functions. We will study how the single-stranded DNA recognition and how the binding effects conformation of the protein and access to the active site. The determinants of recognition and hydrolysis of bleomycin by Gal6 and how they are affected by ssDNA binding will he investigated. We will determine the substrate specificity of Gal6 protease activity, the possible regulatory role of the C-terminal arm, and whether endopeptidase activity is revealed. Finally, we will study the structural basis for tumor cell drug resistance to bleomycin by the human homolog of bleomycin hydrolase.

抗肿瘤药物耐药性是治疗肿瘤的核心问题 人类癌症。半胱氨酸蛋白酶博莱霉素水解酶是第一个 因其水解抗癌药物的能力而受到认可 博来霉素。最近在酵母中发现了这种蛋白酶的同源物 细菌表明这种蛋白质具有保守的细胞功能，然而， 唯一已知的天然底物博莱霉素几乎从未被任何人发现过 这些细胞。 Gal6 是酵母同系物，主要是细胞内的 核蛋白酶，结合 DNA 并在 Gal4 中充当阻遏物 监管体系。这种普遍表达的细胞功能 蛋白质仍然未知。 近年来，人们对细胞内 蛋白酶，因为它们似乎参与重要的监管 流程。博莱霉素水解酶具有多种功能，包括 不寻常的 DNA 结合活性增加了另一个复杂性的维度 这些蛋白酶。酵母博莱霉素水解酶的晶体结构， Gal6，揭示了六聚体结构，其中蛋白酶和 DNA- 结合活动在结构上是相互交织的。该项目的目标 的目的是了解 GaI6 中这两个活动的功能耦合。 这些研究将促进人们对博莱霉素水解酶如何发挥作用的理解 在肿瘤耐药性中发挥作用，并可能提供关于肿瘤耐药性的见解 它的基本细胞功能。我们将研究单链DNA如何 识别以及结合如何影响蛋白质的构象 访问活动站点。识别和水解的决定因素 Gal6 对博莱霉素的影响以及它们如何受到 ssDNA 结合的影响？ 调查了。我们将确定 Gal6 的底物特异性 蛋白酶活性，C 端臂可能的调节作用， 以及是否揭示内肽酶活性。最后我们来学习一下 肿瘤细胞对博莱霉素耐药的结构基础 博莱霉素水解酶的人类同源物。