Non-apoptotic functions of caspase-2 in cell division and genomic stability

项目概要/摘要 几个研究小组已经显示出caspase-2缺乏和加速肿瘤发生之间的强相关性 在淋巴瘤、乳腺癌和肺癌的小鼠模型中。这种表型通常伴随着增强的 细胞增殖和增加的基因组不稳定性，具有最小的可测量的凋亡缺陷。因此 半胱天冬酶-2似乎在维持基因组稳定性中起关键作用，并且可能独立于其作用而起作用 细胞凋亡为了验证这一点，本研究将研究caspase-2在DNA损伤过程中的激活机制。 损伤反应，重点是上游caspase-2调节剂PIDD。核心假设是DNA 损伤诱导两个不同的caspase-2激活平台-一个是PIDD独立的细胞质平台 和需要PIDD的核仁平台-每个平台都提供了不同的底物， 不稳定性通过促凋亡和非凋亡机制。这一假设是根据以下事实提出的： 公开的和在申请人的实验室中产生的初步数据显示，胱天蛋白酶-2在细胞中被激活， 细胞质和核仁中，核仁激活依赖于核仁磷蛋白 核磷蛋白（NPM 1）的组装和功能。这一假设将通过两个具体的 目的：1）确定caspase-2在细胞核和细胞质中的激活机制; 2）鉴定 核仁和胞质复合物对下游caspase-2功能的相对贡献， 它们是如何防止基因组不稳定性。在第一个目标下，一种已经证明的基于成像的方法， 测量半胱天冬酶-2活化，将用于研究不同的半胱天冬酶-2的不同机制， 在细胞核和细胞质中的活化。这些实验将专门探讨PIDD的作用， NPM 1.在第二个目标下，基于初步数据，显示caspase-2缺陷细胞增殖， 更快，积累更多的DNA损伤后复制应力，需要的核仁和 细胞凋亡和非凋亡caspase-2功能的细胞质复合物将在以下背景下进行探索： 细胞凋亡、细胞周期调控、底物裂解和DNA修复机制。方法是创新的 因为它利用了新的基于成像的技术，该技术被设计用于评估单个细胞中caspase-2的激活， 因此，caspase-2激活的定位与细胞凋亡，DNA损伤， 将在每个细胞的基础上直接探索细胞分裂。它还提供了一个新的范例，半胱天冬酶激活， 核仁这项研究是有意义的，因为它提出，位点特异性激活的 细胞质和核仁中的半胱天冬酶-2控制着这种蛋白酶的不同功能， 基因组不稳定性这些机制可能是caspase-2在肿瘤中已知的生理作用的基础 抑制和防止加速老化。最终，这些知识有可能为 如何治疗或预防caspase途径被破坏的疾病。

PROJECT SUMMARY/ABSTRACT Several groups have shown a strong association between caspase-2 deficiency and accelerated tumorigenesis in murine models of lymphoma, breast, and lung cancer. Such phenotypes are often accompanied by enhanced cell proliferation and increased genomic instability with minimal measurable apoptotic defects. Therefore caspase-2 appears to play a crucial role in maintaining genomic stability and may do so independent of its role in apoptosis. To test this, this proposal will study the mechanisms of caspase-2 is activation during the DNA damage response with a focus on the upstream caspase-2 regulator PIDD. The central hypothesis is that DNA damage induces two distinct caspase-2 activation platforms – a cytoplasmic platform that is PIDD independent and a nucleolar platform that requires PIDD – each providing access to distinct substrates that regulate genomic instability by both pro-apoptotic and non-apoptotic mechanisms. This hypothesis has been formulated based on published and preliminary data produced in the applicant’s laboratory showing that caspase-2 is activated in the cytoplasm and in the nucleolus and that the nucleolar activation is dependent on the nucleolar phosphoprotein nucleophosmin (NPM1) for both assembly and function. This hypothesis will be tested by pursuing two specific aims: 1) Determine the mechanisms of caspase-2 activation in the nucleolus versus the cytosol; and 2) Identify the relative contributions of the nucleolar and cytoplasmic complexes to downstream caspase-2 functions and how these protect from genomic instability. Under the first aim, an already proven imaging-based method for measuring caspase-2 activation, will be used to investigate the distinct mechanisms of differential caspase-2 activation in the nucleolus and in the cytosol. These experiments will specifically probe the roles of PIDD and NPM1. Under the second aim, based on preliminary data that shows that caspase-2-deficient cells, proliferate faster and accumulate more DNA damage following replication stress, the requirement of the nucleolar and cytoplasmic complexes for apoptotic and non-apoptotic caspase-2 functions will be explored in the context of apoptosis, cell cycle regulation, substrate cleavage and DNA repair mechanisms. The approach is innovative because it utilizes novel imaging-based techniques that are designed to assess caspase-2 activation in single cells so that the relationship between the localization of caspase-2 activation with apoptosis, DNA damage, and cell division will be directly explored on a per cell basis. It also provides a new paradigm of caspase activation in the nucleolus. The proposed research is significant because it is proposed that site-specific activation of caspase-2 in the cytosol and nucleolus governs distinct functions of this protease that cooperate to protect from genomic instability. These mechanisms may underlie the known physiological roles of caspase-2 in tumor suppression and in protecting against accelerated aging. Ultimately, such knowledge has the potential to inform how diseases where caspase pathways are disrupted can be treated or prevented.