Substrates of SAG-SCF E3 ligase regulate hypoxia response

描述(申请人提供)：已知缺氧可直接或间接地使癌细胞对放化疗产生抗药性，从而导致治疗失败。然而，在低氧癌细胞中，最终导致对当前治疗方法产生耐药性的分子事件还没有很好的定义。我们的长期目标是通过靶向提供这种耐药的分子来诱导缺氧癌细胞的凋亡。凋亡敏感基因SAG是SCF(Skp1、Cullins、F-box蛋白)E3泛素连接酶的环组分。我们发现SAG是在低氧条件下诱导的，当过表达时，在体外和体内都抑制了低氧诱导的细胞凋亡。这项应用的目的是鉴定SAG-SCF E3泛素连接酶底物/靶点，这些底物/靶点的降解有助于耐低氧下的细胞凋亡。中心假说是，低氧诱导SAG与SCF E3泛素连接酶的其他成分形成络合物，促进几种促凋亡蛋白/底物的泛素化和降解，从而抑制低氧诱导的细胞凋亡，作为一种细胞防御/适应性反应。其基本原理是通过鉴定SAG-SCF E3泛素连接酶作为新的缺氧性肿瘤靶点的关键细胞蛋白的降解来阐明连接酶抑制SAG细胞凋亡的机制。验证这一假设的具体目的是：1)鉴定和鉴定SAG-SCF E3泛素连接酶在SAG对缺氧诱导的细胞凋亡的保护中降解的底物；以及2)确定SAG-SCF E3泛素连接酶的选定底物在调节细胞对缺氧和辐射的反应中的作用。通过这项拟议的研究，我们希望鉴定几种SAG-SCF E3连接酶底物，它们的降解有助于在低氧条件下抑制细胞凋亡，从而阐明SAG作用的分子机制，并进一步验证SAG相关的E3连接酶是一个有前途的癌症靶点。此外，确定的底物可以作为开发SAG-SCF E3泛素连接酶特异性抑制剂的生物标志物。这些初步研究将为更详细地研究SAG介导的抑制细胞凋亡的机制奠定基础。

DESCRIPTION (provided by applicant): Hypoxia is known to directly or indirectly confer resistance of cancer cells to radio-and chemo-therapies leading to treatment failure. However, the molecular events that ultimately confer resistance to current therapies in hypoxic cancer cells are not well defined. Our long-range goal is to induce apoptosis of hypoxic cancer cells through targeting the molecules that confer such a resistance. SAG (Sensitive to Apoptosis Gene) is a RING component of SCF (Skpl, Cullins, F-box proteins) E3 ubiquitin ligase. We found that SAG is induced under hypoxia and when over-expressed, inhibits hypoxia-induced apoptosis both in vitro and in vivo. The objective of this application is to identify SAG-SCF E3 ubiquitin ligase substrates/targets whose degradation contributes to apoptosis resistance under hypoxia. The central hypothesis is that hypoxia induces SAG which complexes with other components of SCF E3 ubiquitin ligase to promote the ubiquitination and degradation of several pro-apoptotic proteins/substrates, thus inhibiting hypoxia-induced apoptosis as a cellular defensive/adaptive response. The rationale is to elucidate ligase associated mechanism of SAG apoptosis suppression through identification of critical cellular proteins whose degradation confers such suppression, thus validating SAG-SCF E3 ubiquitin ligase as novel hypoxic cancer targets. Specific aims to test the hypothesis are 1) to identify and characterize substrates of SAG-SCF E3 ubiquitin ligase that are degraded during SAG protection against apoptosis induced by hypoxia; and 2) to define the role of selected substrates of SAG-SCF E3 ubiquitin ligase in regulation of cellular response to hypoxia as well as radiation. Through this proposed research, we expect to identify several SAG-SCF E3 ligase substrates whose degradation contributes to apoptosis inhibition under hypoxia, thus elucidating the molecular mechanism of SAG action and further validating SAG-associated E3 ligase as a promising cancer target. Furthermore, identified substrates can serve as biomarkers for the development of the specific inhibitors of SAG-SCF E3 ubiquitin ligase. These initial studies will set the stage for a more detailed investigation of the mechanism of SAG mediated suppression of apoptosis.