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蛋白酶体是一种能降解P53、Cyclins、NfkappaB、SIC1等重要调控蛋白的特大型(200万Da)多亚单位酶，由18个不同亚基组成的球形调控复合体(RC)与圆柱形20S蛋白酶体的一端或两端相连。在过去的六年中，我们克隆并表达了9个RC亚基。其他几个小组确定了剩下的亚基，今天我们知道了所有18个亚基的序列。虽然20S蛋白酶体的晶体结构已经被解决，但Rc亚基的排列在很大程度上是未知的。由于RC亚基相互之间的位置很重要，我们使用了Far Western blotting和体外组装反应来确定9个亚基之间的亚基之间的接触。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调节复合体中亚基的排列。另一个重要目标是将功能分配给RC亚单位。6个RC亚基属于一个长约400个氨基酸的ATPase家族。虽然ATPase的中心区是高度保守的，但N-端150个残基和C-端序列却是发散的。在构建了一系列嵌合的ATPase之后，我们将确定N-末端和/或C-末端区域是否赋予ATPase不同的功能。几年前，我们发现一个RC亚基(5a)与多泛素链结合。我们最近发现，亚基S2和S4的二聚体也与PolyUb链结合。我们将鉴定这些蛋白质中的PolyUb结合位点(S)。最后，我们发现四个RC亚基可以与20S蛋白酶体亚基的C-末端延伸相对应的多肽交联。我们将研究RC亚基的多肽结合特性，因为一些RC组分可能结合蛋白酶体延伸以组装26S蛋白酶体，而其他组分可能结合蛋白降解底物中的未折叠区域。26S蛋白酶体显然参与了细胞周期的控制，它可能会产生抗原肽，这些抗原肽显示在MHC Class I分子上。由于这些原因，增加对26S蛋白酶体的了解应该具有重大的医学意义。