PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

26S蛋白酶体是一种非常大的（2,000,000 Da）多亚基酶，可降解重要的调节蛋白，如p53， cyclins, NfkappaB， Sic1等。它由一个含有18个不同亚基的球形调节复合体（RC）组成，这些亚基与圆柱形20S蛋白酶体的一端或两端相结合。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下的亚基，今天我们知道所有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。由于定位RC亚基彼此之间的相对位置很重要，我们使用了Far Western blotting和体外组装反应来确定9个亚基之间的亚基接触。我们将继续这些研究，加入尿素中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。另一个重要的目标是将功能分配给RC子单元。6个RC亚基属于一个atp酶家族，长度约为400个氨基酸。虽然ATPases的中心区域是高度保守的，但n端150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）结合多泛素链。我们最近发现一个亚基S2和S4的二聚体也结合polyb链。我们将描述这些蛋白中的polyb结合位点。最后，我们发现四个RC亚基可以交联到对应于20S蛋白酶体亚基c端延伸的肽上。我们将研究RC亚基的肽结合特性，因为一些RC成分可能结合蛋白酶体延伸以组装26S蛋白酶体，而其他成分可能结合蛋白水解底物中的未折叠区域。26S蛋白酶体显然参与控制细胞周期，它可能产生抗原肽，这些抗原肽显示在MHC I类分子上。由于这些原因，增加对26S蛋白酶体的了解应该具有重要的医学意义。

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.