磷酸酶UBLCP1对26S蛋白酶体去磷酸化的机制与功能研究

The 26S proteasome is the primary organelle for protein degradation in eukaryotic cells. It is required for almost every cellular function and is implicated in a variety of human diseases including cancer. However, how this “cellular trashcan” itself is regulated during pathophysiological processes is an important question that remains poorly understood. My earlier work identified the only proteasome-specific phosphatase known to date, UBLCP1, and demonstrated a key role of reversible phosphorylation in proteasome regulation. Subsequenct studies showed that downregulation of UBLCP1 has a significant impact on cancer cell proliferation, migration and signal transduction, suggesting the potential therapeutic value of targeting this proteasome phosphatase. In this proposal, we will systematically examine UBLCP1-mediated proteasome regulation at molecular and cellular levels as well as in the context of cancer formation and progression. To achieve this goal, established approaches of cell biology, biochemistry, mouse genetics and tumor biology will be employed in combination with state-of-the-art research tools such as gene editing with CRISPR/Cas9, phospho-specific antibodies, kinome screen, quantitative mass spectrometry and deep sequencing. Completion of this study will greatly improve our understanding of proteasome regulation as a fundamental question of biology, and will provide new possibilities for proteasome-oriented anti-cancer therapies.

26S蛋白酶体调控几乎所有的细胞功能并与包括癌症在内的多种疾病密切相关。作为真核细胞内负责蛋白质降解的主要细胞器，蛋白酶体自身在生理病理过程中如何被调控是目前亟待解决的重要科学问题。申请人此前报导了目前已知的唯一一个与26S蛋白酶体特异性结合的磷酸酶UBLCP1，揭示了可逆性磷酸化对蛋白酶体的重要调控作用。后续结果显示抑制UBLCP1的表达对癌细胞增殖、迁移、信号转导等有显著影响，提示UBLCP1可能成为新的药物靶标。本项目拟利用细胞生物学、生物化学、动物模型和癌症生物学的成熟实验体系，结合CRISPR基因编辑、磷酸化特异性抗体、全激酶组筛选、定量质谱、深度测序等先进工具方法，对UBLCP1调控蛋白酶体磷酸化的分子机制、细胞功能及其在肿瘤发生发展中的临床意义进行全面系统的研究。对这些问题的解答将加深我们对于蛋白酶体调控这一基本生物学问题的理解，并为针对蛋白酶体系统的抗癌疗法提供新的思路。

26S蛋白酶体负责降解真核细胞中绝大多数蛋白，与几乎所有生命过程及多种疾病密切相关，然而人们对其自身的调控机制还知之甚少。近年来，蛋白酶体磷酸化修饰的生物学意义逐渐被揭示。本项目负责人先前发现了第一个特异性结合蛋白酶体的磷酸酶UBLCP1，对其底物的鉴定和细胞生物学功能的阐明是本项目的主要研究内容。项目取得主要成果如下：.1. 通过体内体外多种实验证明蛋白酶体亚基Rpn1的Ser361是UBLCP1作用的主要磷酸化位点。.2. Rpn1-S361磷酸化在多种细胞和组织类型中广泛存在。阻断该磷酸化修饰导致细胞中蛋白酶体活性降低、细胞增殖及氧化还原稳态均受到影响。.3. 通过对人类全激酶组筛选，鉴定到了Rpn1-S361位点的主要激酶PIM1/2/3。.4. 通过遗传密码拓展技术结合体外生化实验，揭示了Rpn1-S361磷酸化对于蛋白酶体初始组装过程的重要意义。.5. UBLCP1的缺失导致小鼠神经系统发育异常。.上述发现解释了UBLCP1作为蛋白酶体磷酸酶的生物学及生化功能，揭示了一种全新的蛋白酶体调控机制。通过引入非天然氨基酸的方法，第一次从分子层面清晰阐释了磷酸化修饰调控蛋白酶体功能的生化机制。同时，新的蛋白酶体激酶的鉴定也为靶向蛋白酶体的联合用药策略提供了新的可能性。综上，本项目的成果加深和拓展了我们对蛋白酶体磷酸化调控的认识，将本领域的研究推向了新的高度。