Wang Xiaorong, Chen Chi-Fen, Baker Peter R, Chen Phang-lang, Kaiser Peter, Huang Lan
Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560, USA.
Biochemistry. 2007 Mar 20;46(11):3553-65. doi: 10.1021/bi061994u. Epub 2007 Feb 27.
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.
26S蛋白酶体是一种多亚基复合物，负责泛素化底物的降解，在调节各种生物学过程中起关键作用。为了全面了解蛋白酶体复合物的功能和调控机制，一个重要步骤是阐明其亚基组成和翻译后修饰。为实现这一目标，已开发出一种新的亲和纯化策略，使用HB标签的衍生物快速分离人26S蛋白酶体复合物，用于后续的蛋白质组学分析。该复合物的纯化是通过表达HB标签蛋白酶体亚基的稳定293细胞系，以及高亲和力链霉亲和素结合和TEV酶切洗脱来实现的。通过液相色谱-串联质谱法（LC-MS/MS）鉴定了26S蛋白酶体复合物的完整组成，包括最近新确定的亚基。此外，还鉴定了所有已知的蛋白酶体激活蛋白以及参与泛素-蛋白酶体降解途径的成分。除了亚基组成外，还对蛋白酶体亚基的N端修饰和磷酸化进行了表征。已鉴定出8个亚基的12个新磷酸化位点，并确定了25个亚基的N端修饰，其中12个在哺乳动物中以前未被报道。我们还观察到亚基Rpn2的不同N端加工方式，这导致鉴定出该蛋白的两种不同N端。这项工作首次通过亲和纯化和串联质谱对人26S蛋白酶体复合物进行了全面表征。这里获得的详细蛋白质组学分析对于未来旨在全面了解人26S蛋白酶体复合物结构-功能关系的研究具有重要意义。
