PROPERTIES OF AN ATP-UBIQUITIN DEPENDENT PROTEASE

泛素依赖的蛋白水解在细胞周期中起着重要作用 调控 细胞周期蛋白的破坏是细胞完成 有丝分裂和证据最近提出牵连泛素 (Ub)路在这一事件中。 四年前这个实验室鉴定出 纯化的能够降解泛素-溶菌酶缀合物的26 S蛋白酶。 我们提出26 S蛋白酶是由一个已知的20 S蛋白酶组装而成的。 蛋白酶（多催化蛋白酶或MCP）和赋予 ATP依赖性和泛素识别到最终复合物。 这 应用程序涉及这种大ATP的进一步表征， 刺激多亚基降解酶。 分离和测序 26 S蛋白水解颗粒中编码多肽的cDNA将被 持续期间内的 为了研究26 S复合体的组装和拆卸， 制备了完全活性的荧光素化（F）20 S蛋白酶， 表明它们可以容易地组装成活性26 S颗粒。 我们将 继续检查装配/拆卸，具体目的是 测试蛋白酶机制的“核糖体”与“垃圾处理”模型。 F-标记的20 S蛋白酶也将用于确定其是否为 亚基在体外和体内均处于动态平衡状态 培养细胞。 使用光交联方法，我们鉴定了两个26 S 结合ATP的亚基。 将26 S蛋白酶与γ-32 PO 4- ATP产生三个磷酸化亚基。 其中一个是32 K亚基 我们有证据表明另一种是一种新的蛋白质 26 S颗粒固有的激酶。 磷酸化在 蛋白酶活性将进一步研究。 我们还将使用照片- 交联以鉴定结合泛素的26 S组分。 双泛素 在赖氨酸48处通过异肽键连接的分子将被衍生化， 与26 S络合物光交联。 两年前，我们开发了一种快速， 一种非常便宜的合成肽类如泛素羧基的方法 extensions. 这项技术将用于制备链特异性 26 S复合物中组分的抗体，并制备栓系的 用于探测20 S蛋白酶中的活性位点的底物。 最后，两个 精确地将pentaUb加工成Ub的单独酶已经被 鉴定 我们会提纯每一种酶， 对多聚泛素代谢的影响

Ubiquitin-dependent proteolysis plays a fundamental role in cell cycle regulation. Destruction of cyclin is required for cells to complete mitosis and evidence has recently been presented implicating the ubiquitin (Ub) pathway in this event. Four years ago this lab identified and purified a 26S protease capable of degrading ubiquitin-lysozyme conjugates. We proposed that the 26S protease was assembled from a well-known 20S protease (the multicatalytic protease or MCP) and polypeptides that confer ATP-dependence and ubiquitin recognition to the final complex. This application deals with the further characterization of this large ATP- stimulated multisubunit degradative enzyme. The isolation and sequencing of cDNAs that encode polypeptides in the 26S proteolytic particle will be continued. To study the assembly and disassembly of the 26S complex we have prepared fully active, fluoresceinated (F) 20S proteases and have shown that they can readily assemble into active 26S particles. We will continue to examine assembly/disassembly with the specific intention of testing "ribosome" versus "garbage disposal" models for protease mechanism. F-labeled 20S protease will also be employed to determine whether its subunits are in a state of dynamic equilibrium both in vitro and in living culture cells. Using photo-crosslinking approaches we identified two 26S subunits that bind ATP. Incubation of the 26S protease with gamma-32PO4- ATP produced three phosphorylated subunits. One of these is a 32K subunit in the 20S protease; we have evidence that another is a novel protein kinase inherent to the 26S particle. The role of phosphorylation in protease activity will be studied further. We will also use photo- crosslinking to identify 26S components that bind ubiquitin. Diubiquitin molecules linked by an isopeptide bond at lysine 48 will be derivatized and photocrosslinked to the 26S complex. Two years ago we developed a fast and very cheap method for synthesizing peptides as ubiquitin carboxyl extensions. This technology will be used to prepare chain-specific antibodies to components in the 26S complex and to prepare tethered substrates for probing active sites in the 20S protease. Finally, two separate enzymes that accurately process pentaUb to Ub have been identified. We will purify each enzyme, and we will continue our studies on poly-ubiquitin metabolism.