PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE

26 S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、Sic 1等。它由一个球形调节复合物（RC）组成，含有18个不同的亚基，与圆柱形20 S蛋白酶体的一端或两端相关。 在过去的六年中，我们克隆并表达了9个RC亚基。 其他几个研究小组鉴定了剩下的亚基，今天我们知道了所有18个亚基的序列。 虽然20 S蛋白酶体的晶体结构已经被解决，RC亚基的排列在很大程度上是未知的。 因为它是重要的位置RC亚基相对于彼此，我们已经使用远Western印迹和体外组装反应，以确定9个亚基之间的亚基间的接触。 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，以分析调节复合物中亚基的排列。 另一个重要目标是为RC子单元分配功能。 六个RC亚基属于长度约400个氨基酸的ATP酶家族。 虽然ATP酶的中心区域是高度保守的，但N-末端150个残基和C-末端序列是不同的。 在构建了一系列嵌合ATP酶之后，我们将确定N-末端和/或C-末端区域是否赋予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polyUb链。 我们将表征这些蛋白质内的polyUb结合位点。最后，我们发现，四个RC亚基可以交联到一个肽对应的20 S蛋白酶体亚基的C-末端延伸。 我们将研究RC亚基的肽结合特性，因为一些RC组分可能结合蛋白酶体延伸以组装26 S蛋白酶体，而其他组分可能结合蛋白水解底物中的未折叠区域。 26 S蛋白酶体明显参与细胞周期的控制，并且它可能产生在MHC I类分子上展示的抗原肽。 基于这些原因，增加对26 S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.