The role of p53 redox-modification in cell fate signaling

描述(由申请人提供)：肿瘤抑制基因P53是控制细胞增殖和细胞命运决定的多种癌症相关信号网络的中心枢纽。然而， 我们对p53了解得越多，它的作用就变得越复杂和动态。最近的研究表明，P53参与了一系列日益多样化的细胞命运，除了细胞凋亡外，还包括坏死和衰老。P53显著的生物化学复杂性是其多种生理功能所必需的，然而我们对这些机制的了解还远未完成。例如，数十个实验室的研究表明，就细胞命运信号而言，非核P53功能几乎与DNA反式激活一样重要。事实上，即使在没有功能DNA结合域的情况下，在没有RNA合成、蛋白质翻译的情况下，甚至在没有核的情况下，P53也可以通过转录无关的机制诱导细胞凋亡。然而，与经典的p53 DNA反式激活反应相比，这一过程的机制细节仍然很少被描述，值得进一步研究。氧化还原反应是许多促和抗细胞凋亡的细胞程序中必不可少的生化过程，这些过程指导着癌细胞的新陈代谢、肿瘤生长、血管生成和转移。这些反应的特异性主要是由所选蛋白质中反应性巯基的高度氧化性所决定的。P53是一个含有10个半胱氨酸的锌指蛋白，都在其DNA结合区内，氧化还原调节被广泛推测在其调控中发挥作用。然而，由于技术的限制，几乎所有评估P53氧化还原调节和功能的研究都是研究分离的重组蛋白。几乎没有证据表明，在与生理相关的条件或压力下，P53在其半胱氨酸上的细胞中受到氧化还原调节。使用一种新的基于质谱学的氧化还原分析技术OxMRM以及非还原的SDS-PAGE，我们发现p53在细胞环境中受到氧化还原调节，以响应DNA损伤和氧化应激。这些结果以及我们的生化和功能研究表明，P53的氧化还原调节是细胞命运信号转导中的关键步骤，包括细胞凋亡、衰老和坏死。这一建议将进一步表征P53的氧化还原调节，包括1)鉴定二硫键连接的P53配对，2)量化和确定P53在线粒体上的氧化还原状态，以及3)确定转录非依赖的P53信号转导阶段，其中氧化还原调节是必不可少的。从长远来看，氧化还原动态平衡的改变是癌变的一个标志，并可能影响P53的功能。例如，解除对P53氧化还原调节的调控是DNA损伤细胞逃避凋亡并推动肿瘤发生的潜在机制。

DESCRIPTION (provided by applicant): The tumor suppressor p53 is a central hub in multiple cancer-related signaling networks that control cell proliferation and cell fate decisions. However, the more we learn about p53 the more complex and dynamic its role becomes. Recent studies have revealed that p53 is involved in an increasingly diverse range of cellular fates, including necrosis and senescence in addition to apoptosis. The remarkable biochemical complexity of p53 is essential to its diverse physiological roles, yet our understanding of these mechanisms is far from complete. For example, as shown in studies by dozens of laboratories, non-nuclear p53 functions are nearly as important as DNA transactivation with regard to cell fate signaling. In fact, p53 can induce apoptosis in cells through transcription-independent mechanisms even without a functional DNA binding domain, in the absence of RNA synthesis, protein translation, or even without a nucleus. However, the mechanistic details of this process remains much less characterized than classical p53 DNA transactivation response and warrants further investigation. Oxidation-reduction (redox) reactions are essential biochemical processes in many pro- and anti-apoptotic cellular programs that direct cancer cell metabolism, tumor growth, angiogenesis and metastasis. The specificity of these reactions is primarily defined by the high oxidizability of reactive sulfhydryls in select proteins. p53 is a zinc finger protein with ten cysteines, all within its DNA binding domain, and redox-regulated is widely speculated to play a role in its regulation. However, due to technological limitations, nearly all of the studies assessing p53 redox regulation and function have investigated isolated, recombinant protein. There is very little evidence that p53 is redox-regulated in cells on one of its cysteines under physiologically-relevant conditions or stresses. Using a novel mass spectrometry-based redox analysis technology, OxMRM, as well as non-reducing SDS-PAGE, we've discovered that p53 is redox-regulated in a cellular context in response to DNA damage and oxidative stress. These results, along with our biochemical and functional studies, suggest that redox- regulation of p53 is a critical step in cell fate signaling including apoptosis, senescence, and necrosis. This proposal will further characterize the redox-regulation of p53, including 1) identification of the disulfide-linked p53 partner, 2) quantify and define the redox-status of p53 at the mitochondria, and 3) determine the stage of transcription-independent p53 signaling in which redox-regulation is essential. In the long term, altered redox homeostasis is a hallmark of carcinogenesis and may impact p53 function. For example, deregulation of p53 redox-regulation is a potential mechanism by which DNA damaged cells could evade apoptosis and drive tumorigenesis.