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

• 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
• 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

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-核糖基化是由17个酶的家族合成的翻译后蛋白质修饰，称为PARPS（Poly（ADP-核糖）聚合酶）。人类细胞表达17种不同的PARP蛋白；许多人的功能和修改目标尚未确定。对PARP家族的更深入的了解在临床上很重要，因为PARP是针对PARP-1（PARP家族的创始成员）的高度可药酶和PARP抑制剂，目前正在超过14个临床试验中，用于治疗乳腺，卵巢，肺，肺，肺，Skin和结直肠癌。 PARPS的宏亚家族（PARP-9，-14和-15）最初在蛋白质的筛选中鉴定出其在B细胞淋巴瘤细胞中的过表达导致细胞迁移率的提高，并且PARP-9和PARP-14此后已将其与癌细胞迁移的作用联系起来。尽管这些早期分析表明宏PARP在细胞运动的调节中的功能，但该调节的机制尚不清楚。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，以及适当的细胞运动所需的小GTPase活性的缺陷，RhoA。拟议的研究的目的是确定有助于调节RhoA信号传导和细胞运动性的PARP-9和PARP-14活性的目标和机制。第一个提出的实验目的检验了以下假设：PARP -9和-14直接有助于对人类细胞中RhoA活性的适当时空控制。为此，将使用RhoA的本地化和活动状态在主动迁移细胞中的主动化和活动状态。 将测试基于FRET的记者，以及PARP-9和PARP-14耗竭对这种时空活性的影响。与RhoA活性部位有关，也将实时观察PARP -9和-14的定位。最后，如果RhoA ISA的直接目标ADP-核糖基化靶标，以及PARP-9或PARP-14是否有助于此修饰，则将直接测试它。第二个实验目的将通过测试各种PARP-9和PARP-14突变体营救观察到的耗竭表型的能力，确定适当细胞运动和膜动力学所需的PARP-9和PARP-14的活性和域。第三个目标将采用生化方法来识别PARP-9和-14结合伙伴以及ADP-核糖基化靶标，可能有助于细胞运动和RhoA调节。还将探索潜在靶蛋白的ADP-核糖修饰有助于细胞运动和膜动力学。总之，这些实验将进一步了解人类细胞中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，失调的细胞运动有助于转移，针对细胞运动机制的小分子抑制剂可降低浸润并抑制肿瘤的生长。因此，将来，针对细胞运动中PARP-9和PARP-14功能的药物可能是转移性癌症的有前途的治疗方法。 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，都要求体内的单个细胞快速和响应地从一个位置迁移到另一个位置。这种受控的迁移行为中的缺陷可能有助于转移性癌症的发展；因此，了解细胞迁移的蛋白质和涉及的信号对于了解正常过程和疾病过程至关重要。本应用程序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的新作用，在控制细胞迁移中，从而扩展了我们对细胞如何指导其运动的理解，并阐明了两种蛋白质，这些蛋白质可用于阻止转移性癌细胞的扩散。

项目成果

A systematic analysis of the PARP protein family identifies new functions critical for cell physiology.

• DOI: 10.1038/ncomms3240
• 发表时间: 2013
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Vyas, Sejal;Chesarone-Cataldo, Melissa;Todorova, Tanya;Huang, Yun-Han;Chang, Paul
• 通讯作者: Chang, Paul