Mcl-1对凋亡调控、胚胎发育、体内稳态的维持等有至关重要的作用。Mcl-1表达和调节异常会导致各种疾病，特别是引发各种肿瘤。因而，Mcl-1 可作为肿瘤治疗的靶点。鉴于Mcl-1 的生理功能重要,调节Mcl-1的表达水平是一种控制肿瘤发生发展的治疗策略。因而，深入了解Mcl-1 蛋白稳定性的调控机制, 将有助于阐明因其表达异常而诱发肿瘤的根本原因。调控Mcl-1蛋白稳定性的途径多样，机制复杂。据报道，Mule, Fbw7, βTrCP等泛素化激酶能促进Mcl-1降解。但是，我们发现,Mcl-1在细胞内的基本代谢不依赖于泛素化降解途径，具体分子机制不详。因而，应用siRNA高通量筛选的方法找到影响Mcl-1蛋白基本代谢的调节因子，阐明Mcl-1基本代谢的分子机制是本课题的目的。同时，我们将评价这些调节因子在Mcl-1 依赖型的肿瘤细胞中的功能和作用，为发展新的肿瘤治疗策略提供理论依据。

Impaired apoptosis is a hallmark of most cancers. In addition to promoting cellular transformation, defective apoptosis also often blunts the response to standard-of-care chemotherapeutics that are used in the clinic. Thus, restoring normal apoptotic responses in cancer cells by targeting survival factors such as Mcl-1 is an attractive approach to treating cancer and overcoming chemoresistance. Here, we will focus on targeting the pathways that control Mcl-1 protein stability to modulate its pro-survival function. Normaly, Mcl-1 is a very labile protein, with a half-life of less than an hour in most cells. It's stability is even further reduced when ubiquitinylated by one of several reported E3 ligases-Mule,Fbw7,βTrCP-whereupon it is degraded by the proteasome.Which of the ligases modify Mcl-1 appears highly context-specific; for example, Fbw7 drives Mcl-1 degradation principally during mitotic arrest. Thus, the stability of Mcl-1 appears to be regulated by multiple pathways. While it is clear that the reported E3 ligases can drive Mcl-1 degradation in specific contexts, our studies have thus far excluded a specific role Mule, Fbw7, and βTrCP in controlling the steady-state basal turnover of Mcl-1. Our results also suggest that the lysines of Mcl-1 are not required for its basal turnover, although it does remain proteasome-dependent. We conclude that the basal turnover of Mcl-1 must proceed by a mechanism that has not been fully characterized. Given the background and compelling preliminary data to hand, we propose to test the following hypotheses:Aim 1 That the control of basal Mcl-1 turnover is mediated by a novel ubiquitin-independent mechanism;Aim 2 That manipulating these pathways in tumor cells dependent on Mcl-1 for their survival will cause outright cytotoxicity and/or modulate their response to known standard-of-care chemotherapeutic agents. To address these aims we have prepared a system to screen genetically for factors that mediate the basal turnover of Mcl-1. We have engineered a Mcl-1 construct fused to a luciferase reporter. This fusion protein maintains normal Mcl-1 pro-survival activity and its stability is controlled analogously to endogenous Mcl-1. Cell lines that stably express the Mcl-1 luciferase reporter have been prepared and conditions established for RNAi screening. To identify regulators of basal turnover we will screen for siRNAs that inhibit the rapid reduction of reporter activity upon treatment with cycloheximide. This screen will be performed on a genome-wide scale as the underlying mechanism is unclear. To functionally validate hits we will manipulate the pathways identified using siRNA and/or shRNA and/or chemical tools in tumour cell lines known to require Mcl-1 for their survival. We will measure the impact on cell viability of trageting these pathways alone and in combination with chemotherapeutic agents.

Mcl-1 对凋亡调控、胚胎发育、体内稳态的维持等有至关重要的作用。Mcl-1 表达和调节异常会导致各种疾病，特别是引发各种肿瘤。因而，Mcl-1 可作为肿瘤治疗的靶点。鉴于 Mcl-1的生理功能重要,调节 Mcl-1 的表达水平是一种控制肿瘤发生发展的治疗策略。因而，深入了解 Mcl-1 蛋白稳定性的调控机制, 将有助于阐明因其表达异常而诱发肿瘤的根本原因。调控 Mcl-1 蛋白稳定性的途径多样，机制复杂。据报道，Mule, Fbw7, β TrCP 等泛素化激酶能促进 Mcl-1 降解。但是，我们发现,Mcl-1 在细胞内的基本代谢不依赖于泛素化降解途径，具体分子机制不详。因而，应用 siRNA 高通量筛选的方法找到影响 Mcl-1 蛋白基本代谢的调节因子，阐明 Mcl-1 基本代谢的分子机制是本课题的目的。同时，我们将评价这些调节因子在Mcl-1 依赖型的肿瘤细胞中的功能和作用，为发展新的肿瘤治疗策略提供理论依据。通过应用siRNA 高通量筛选得到候选基因后，我们从得分较高的候选基因中鉴别出了两个影响Mcl-1蛋白基本代谢的调节因子。后续工作，我们将采用基因敲除的手段分别在细胞系和小鼠体内深入研究这两个基因对Mcl-1蛋白的稳定性影响。与此同时，我们也并行探讨了其他研究课题，包括：1.探讨老年痴呆、帕金森症等神经性疾病发病过程中细胞凋亡的分子机理；2.探讨大剂量激光辐照（high-fluence low-power laser irradiation, HF-LPLI) 治疗肿瘤的免疫应答机制；3.探讨低剂量激光疗法（low-level laser therapy，LLLT）对于治疗2型糖尿病、阿尔茨海默尔症、帕金森症等重大慢性疾病的分子机制；4.探讨低剂量激光疗法对于提高机体免疫的机制。研究所取得的进展和成果将在报告正文中详细阐述。