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

项目摘要/摘要 有几个研究小组表明，caspase-2缺陷与加速肿瘤的形成有很强的相关性。 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 细胞增殖和基因组的不稳定性增加，并伴有最小的可测量的凋亡缺陷。因此 Caspase-2似乎在维持基因组稳定性方面起着至关重要的作用，而且这种作用可能与其作用无关 在细胞凋亡中。为了验证这一点，这项提议将研究caspase-2在DNA过程中的激活机制 损伤反应，重点是上游的caspase-2调节因子PIDD。中心假设是DNA 损伤诱导两个不同的caspase-2激活平台--一个不依赖于PIDD的细胞质平台 以及需要PIDD的核仁平台--每个都提供对调节基因组的不同底物的通道 既有促凋亡机制又有非凋亡机制的不稳定性。这一假设是基于以下假设提出的 申请人实验室公布的初步数据显示，caspase-2在 核仁的激活依赖于核仁磷蛋白。 核磷脂(NPM1)对组装和功能都有作用。这一假设将通过追求两个具体的 目的：1)确定caspase-2在核仁和胞浆中的激活机制；以及2)确定 核仁和细胞质复合体对caspase-2下游功能的相对贡献 它们是如何防止基因组不稳定的。在第一个目标下，已经证明了一种基于成像的方法 测量caspase-2的活性，将被用于研究差异caspase-2的不同机制 核仁和胞浆中的激活。这些实验将具体探索PIDD和PIDD的作用 NPM1。在第二个目标下，基于初步数据，caspase-2缺失的细胞增殖 复制应激后更快并积累更多的DNA损伤，核仁和 凋亡和非凋亡caspase-2功能的细胞质复合体将在以下背景下探索 细胞凋亡、细胞周期调控、底物切割和DNA修复机制。这种方法是创新的 因为它使用了新的基于成像的技术，这些技术被设计成在单个 从而使caspase-2激活的定位与细胞凋亡、DNA损伤以及 将在每个细胞的基础上直接探索细胞分裂。它还提供了一种新的caspase激活范例 核仁。这项拟议的研究具有重要意义，因为它提出了位点特异性激活 胞浆和核仁中的caspase-2调控这种蛋白水解酶的不同功能，它们协同保护 基因组不稳定。这些机制可能是caspase-2在肿瘤中已知的生理作用的基础。 抑制和防止加速衰老。归根结底，这种知识有可能为 如何治疗或预防半胱氨酸天冬氨酸酶途径被破坏的疾病。

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.