Roles for Human PARPs in Regulating the Cytoskeleton and Cell Motility

人类 PARP 在调节细胞骨架和细胞运动中的作用

基本信息

批准号：
8395494
负责人：
Melissa Chesarone Cataldo
金额：
$ 1.52万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2012-11-08
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8395494
关键词：
ADP ribosylation Actins Adenosine Diphosphate Ribose Amino Acids B-Cell Lymphomas Behavior Binding Biochemical Breast Catalytic Domain Cell membrane Cell physiology Cells Clinical Treatment Clinical Trials Colorectal Cancer Coupled Cytoskeleton Data Defect Development Disease Disseminated Malignant Neoplasm Drug Delivery Systems Embryonic Development Enzymes Family Fluorescence Resonance Energy Transfer Focal Adhesions Future Guanosine Triphosphate Phosphohydrolases Human Immune response Individual Link Location Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of ovary Mediating Membrane Microfilaments Modification Monomeric GTP-Binding Proteins Movement Neoplasm Metastasis Normal Cell Pathway interactions Pharmacologic Substance Phenotype Phosphorylation Physiological Processes Play Poly(ADP-ribose) Polymerases Post-Translational Protein Processing Process Proteins Regulation Reporter Research Research Training Role Side Signal Transduction Signaling Protein Site Site-Directed Mutagenesis Skin Cancer Somatic Cell Testing Therapeutic Time Training Programs Ubiquitination base cancer cell cell cortex cell motility design inhibitor/antagonist insight member mutant novel protein function research study rho GTP-Binding Proteins small molecule spatiotemporal tumor growth tumor progression wound

项目摘要

DESCRIPTION (provided by applicant): ADP-ribosylation is a post-translational protein modification synthesized by a family of 17 enzymes, called PARPs (poly(ADP-ribose) polymerases). Human cells express 17 different PARP proteins; the functions and modification targets of many have not yet been identified. Deeper understanding of the PARP family is clinically important, as PARPs are highly druggable enzymes and PARP inhibitors directed against PARP-1, the founding member of the PARP family, are currently in over 14 clinical trials for the treatments of breast, ovarian, lung, skin and colorectal cancers. The Macro subfamily of PARPs (PARP-9, -14 and -15) was initially identified in a screen for proteins whose over-expression in B-cell lymphoma cells resulted in increased cell migration rates, and PARP-9 and PARP-14 have since been linked to roles in cancer cell migration. While these early analyses suggested functions for the Macro PARPs in the regulation of cell motility, the mechanism of this regulation has remained unclear. Recently, we have found that depletion of either PARP-9 or PARP-14 from human somatic cells causes severe defects in cell motility and membrane dynamics, along with defects in the activity of a small GTPase required for proper cell motility, RhoA. The objective of the proposed research is to determine both the targets and the mechanisms of PARP-9 and PARP-14 activity that contribute to the regulation of RhoA signaling and cell motility. The first proposed experimental aim tests the hypothesis that PARP-9 and -14 directly contribute to the proper spatiotemporal control of RhoA activity in human cells. To this end, the localization and activity state of RhoA in actively migrating cells will be examined using FRET-based reporters, and the effects of PARP-9 and PARP-14 depletion on this spatiotemporal activity will be tested. The localization of PARP-9 and -14 will also be observed, in real time, in relation to sites of RhoA activity. Finally, it will be directly tested if RhoA isa direct target of ADP-ribosylation, and if PARP-9 or PARP-14 contributes to this modification. The second experimental aim will determine the activity and the domains of PARP-9 and PARP-14 required for proper cell motility and membrane dynamics, by testing the ability of various PARP-9 and PARP-14 mutants to rescue the observed depletion phenotypes. The third aim will take a biochemical approach to identify PARP-9 and -14 binding partners and ADP-ribosylation targets that may contribute to cell motility and RhoA regulation. How ADP-ribose modification of potential target proteins contributes to cell motility and membrane dynamics will also be explored. Together, these experiments will further our understanding of PARP protein function in human cells and will describe a novel role for PARPs in cell migration and RhoA regulation. Importantly, deregulated cell motility contributes to metastasis, and small molecule inhibitors targeting the cell motility machinery decrease invasion and suppress tumor growth. Thus in the future, drugs targeting PARP-9 and PARP-14 functions in cell motility may be promising therapeutic treatments for metastatic cancers. PUBLIC HEALTH RELEVANCE: Many physiological processes, including wound closure, embryonic development and immune responses, require individual cells within the body to rapidly and responsively migrate from one location to another. Defects in this controlled migratory behavior can contribute to the development of metastatic cancers; understanding the proteins and the signals involved in cell migration is therefore critical to understanding both normal and disease processes. The research training program described in this application will explore and define a novel role for two proteins, PARP-9 and PARP-14, in the control of cell migration, thereby extending our understanding of how cells direct their movements, and elucidating two proteins that can be targeted to stop the spread of metastatic cancer cells.

描述（由申请人提供）：ADP-核糖基化是一种翻译后蛋白质修饰，由称为 PARP（聚（ADP-核糖）聚合酶）的 17 种酶家族合成。人类细胞表达 17 种不同的 PARP 蛋白；许多功能和修改目标尚未确定。对 PARP 家族的深入了解在临床上非常重要，因为 PARP 是高度可成药的酶，而针对 PARP 家族创始成员 PARP-1 的 PARP 抑制剂目前正在进行超过 14 项临床试验，用于治疗乳腺癌、卵巢癌、肺癌、皮肤癌和结直肠癌。 PARP 宏亚家族（PARP-9、-14 和 -15）最初是在筛选蛋白质时发现的，这些蛋白质在 B 细胞淋巴瘤细胞中过度表达导致细胞迁移率增加，PARP-9 和 PARP-14 已自从被认为与癌细胞迁移的作用有关。虽然这些早期分析表明宏 PARP 在细胞运动调节中发挥作用，但这种调节的机制仍不清楚。最近，我们发现人类体细胞中 PARP-9 或 PARP-14 的耗竭会导致细胞运动和膜动力学的严重缺陷，以及适当细胞运动所需的小 GTP 酶 RhoA 的活性缺陷。本研究的目的是确定 PARP-9 和 PARP-14 活性的靶点和机制，这些活性有助于调节 RhoA 信号传导和细胞运动。第一个提出的实验目标测试了以下假设：PARP-9 和-14 直接有助于人类细胞中 RhoA 活性的适当时空控制。为此，将使用以下方法检查活跃迁移细胞中 RhoA 的定位和活性状态基于 FRET 的报告基因，以及 PARP-9 和 PARP-14 消耗对此时空活动的影响将得到测试。还将实时观察与 RhoA 活性位点相关的 PARP-9 和 -14 的定位。最后，将直接测试 RhoA 是否是 ADP-核糖基化的直接目标，以及 PARP-9 或 PARP-14 是否有助于这种修饰。第二个实验目标将通过测试各种 PARP-9 和 PARP-14 突变体拯救观察到的耗尽表型的能力，确定适当细胞运动和膜动力学所需的 PARP-9 和 PARP-14 的活性和结构域。第三个目标将采用生化方法来识别可能有助于细胞运动和 RhoA 调节的 PARP-9 和 -14 结合伴侣以及 ADP-核糖基化靶标。还将探讨潜在靶蛋白的 ADP-核糖修饰如何促进细胞运动和膜动力学。总之，这些实验将进一步加深我们对人类细胞中 PARP 蛋白功能的理解，并将描述 PARP 在细胞迁移和 RhoA 调节中的新作用。重要的是，细胞运动失调有助于转移，而针对细胞运动机制的小分子抑制剂可减少侵袭并抑制肿瘤生长。因此，在未来，针对细胞运动中 PARP-9 和 PARP-14 功能的药物可能成为治疗转移性癌症的有希望的治疗方法。公共卫生相关性：许多生理过程，包括伤口闭合、胚胎发育和免疫反应，都需要体内的单个细胞快速、灵敏地从一个位置迁移到另一个位置。这种受控迁移行为的缺陷可能会导致转移性癌症的发展。因此，了解细胞迁移所涉及的蛋白质和信号对于了解正常和疾病过程至关重要。本申请中描述的研究培训计划将探索和定义两种蛋白质（PARP-9 和 PARP-14）在控制细胞迁移中的新作用，从而扩展我们对细胞如何指导其运动的理解，并阐明两种蛋白质可以有针对性地阻止转移性癌细胞的扩散。