The role of p53 redox-modification in cell fate signaling

描述（由申请人提供）：肿瘤抑制因子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，在这方面， 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，p53参与了越来越多的细胞命运，包括坏死和衰老以及凋亡。p53的显著的生化复杂性对其多种生理作用至关重要，但我们对这些机制的理解还远未完成。例如，正如几十个实验室的研究所示，非核p53功能在细胞命运信号传导方面几乎与DNA反式激活一样重要。事实上，即使没有功能性DNA结合结构域，没有RNA合成，蛋白质翻译，甚至没有细胞核，p53也可以通过转录非依赖性机制诱导细胞凋亡。然而，这一过程的机制细节仍然远不如经典的p53 DNA反式激活反应的特点，值得进一步调查。 氧化还原反应是许多促凋亡和抗凋亡细胞程序中必不可少的生化过程，这些程序指导癌细胞代谢、肿瘤生长、血管生成和转移。这些反应的特异性主要由所选蛋白质中反应性巯基的高氧化性来定义。p53是一种具有10个半胱氨酸的锌指蛋白，所有这些半胱氨酸都在其DNA结合结构域内，并且氧化还原调节被广泛推测在其调节中起作用。然而，由于技术限制，几乎所有评估p53氧化还原调节和功能的研究都研究了分离的重组蛋白。很少有证据表明，p53在生理相关条件或应激下在细胞中的一个半胱氨酸上被氧化还原调节。 使用一种新的基于质谱的氧化还原分析技术，OxMRM，以及非还原性SDS-PAGE，我们发现p53在细胞环境中响应DNA损伤和氧化应激而受到氧化还原调节。这些结果，沿着我们的生物化学和功能研究，提示p53的氧化还原调节是细胞命运信号传导（包括凋亡、衰老和坏死）中的关键步骤。该提案将进一步表征p53的氧化还原调节，包括1）鉴定二硫键连接的p53伴侣，2）定量和定义线粒体中p53的氧化还原状态，以及3）确定氧化还原调节至关重要的转录非依赖性p53信号传导的阶段。从长远来看，改变氧化还原稳态是致癌的标志，并可能影响p53功能。例如，p53氧化还原调节的失调是DNA损伤细胞逃避凋亡和驱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.