PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

ATP/泛素依赖性蛋白酶的特性

• 批准号: 6329672
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 32.37万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329672
• 关键词: active sites; adenosine triphosphate; adenosinetriphosphatase; antibody; binding sites; complementary DNA; crosslink; electron microscopy; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; hybrid enzyme; hydropathy; laboratory rabbit; mass spectrometry; peptidases; proteasome; protein structure function; proteolysis; tissue /cell culture; ubiquitin; western blottings

The 26S proteasome is an extremely large (2,000,000 Da) multisubunit enzyme that degrades important regulatory proteins such as p53, cyclins, NfkappaB, Sic1, etc. It is comprised of a spherical regulatory complex (RC) containing 18 different subunits that associates with one or both ends of the cylindrical 20S proteasome. Over the past six years, we cloned and expressed 9 of the RC subunits. Several other groups identified the remaining subunits, and today we know the sequences of all 18 subunits. Although the crystal structure of the 20S proteasome has been solved, the arrangement of RC subunits is largely unknown. Because it is important to position the RC subunits relative to one another, we have used Far Western blotting and in vitro assembly reactions to determine intersubunit contacts among 9 of the subunits. We will continue these studies adding limited dissociation of the RC in urea and mass spectrometry to analyze the arrangement of subunits in the regulatory complex. Another important goal is to assign functions to the RC subunits. Six RC subunits belong to a family of ATPases approximately 400 amino acids in length. Although the central regions in the ATPases are highly conserved, the N-terminal 150 residues and C-terminal sequences are divergent. Having constructed a series of chimeric ATPases we will determine whether the N-terminal and/or C- terminal regions confer distinct functions to the ATPases. Several years ago we found that one RC subunit (5a) binds polyubiquitin chains. We recently discovered that a dimer of subunits S2 and S4 also binds polyUb chains. We will characterize the polyUb binding site(s) within these proteins. Finally, we have found that four RC subunits can be crosslinked to a peptide corresponding to a C-terminal extension of a 20S proteasome subunit. We will examine the peptide binding properties of RC subunits because some RC components may bind proteasome extensions to assemble the 26S proteasome and others may bind unfolded regions in proteolytic substrates. The 26S proteaseome is clearly involved in control of the cell cycle, and it likely generates antigenic peptides that are displayed on MHC Class I molecules. For these reasons, increased knowledge of the 26S proteasome should have significant medical implications.

26S 蛋白酶体是一种非常大 (2,000,000 Da) 的多亚基酶，可降解重要的调节蛋白，如 p53、细胞周期蛋白、NfkappaB、Sic1 等。它由含有 18 个不同亚基的球形调节复合物 (RC) 组成，与圆柱形 20S 蛋白酶体的一端或两端相连。 在过去的六年里，我们克隆并表达了 9 个 RC 亚基。 其他几个小组鉴定了剩余的亚基，今天我们知道了所有 18 个亚基的序列。 尽管20S蛋白酶体的晶体结构已被解析，但RC亚基的排列很大程度上未知。 由于将 RC 亚基彼此相对定位很重要，因此我们使用 Far Western 印迹和体外组装反应来确定 9 个亚基之间的亚基间接触。 我们将继续这些研究，增加尿素中 RC 的有限解离和质谱分析，以分析调节复合物中亚基的排列。 另一个重要目标是将功能分配给 RC 子单元。 六个 RC 亚基属于长度约为 400 个氨基酸的 ATP 酶家族。 尽管 ATP 酶的中心区域高度保守，但 N 端 150 个残基和 C 端序列是不同的。 构建了一系列嵌合 ATP 酶后，我们将确定 N 端和/或 C 端区域是否赋予 ATP 酶不同的功能。几年前，我们发现一个 RC 亚基 (5a) 结合多聚泛素链。 我们最近发现亚基 S2 和 S4 的二聚体也结合多聚泛素链。 我们将表征这些蛋白质内的多聚泛素结合位点。最后，我们发现四个 RC 亚基可以与对应于 20S 蛋白酶体亚基 C 端延伸的肽交联。 我们将检查 RC 亚基的肽结合特性，因为一些 RC 成分可能会结合蛋白酶体延伸以组装 26S 蛋白酶体，而其他成分可能会结合蛋白水解底物中的未折叠区域。 26S 蛋白酶组显然参与细胞周期的控制，并且它可能产生展示在 MHC I 类分子上的抗原肽。 由于这些原因，增加对 26S 蛋白酶体的了解应该具有重要的医学意义。