Significance of Caspase-Mediated Cleavage of PARP-1 in Cell Death

Caspase 介导的 PARP-1 裂解在细胞死亡中的意义

• 批准号: 7387068
• 负责人: Karen Kate David
• 金额: $ 3.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7387068
• 关键词: Affect; Aging; Animal Model; Apoptosis; Apoptotic; Biochemical; Biological; Brain Injuries; Caspase; Cell Death; Cell physiology; Cells; Cessation of life; Cleaved cell; Condition; DNA Damage; DNA Repair; Development; Disease; Disease Progression; Event; Experimental Models; Fibroblasts; Glutamates; Homeostasis; Injury; Intervention; Knock-in Mouse; Mediating; Mus; Mutate; Mutation; Myocardial Infarction; Nerve Degeneration; Neurons; Organ; Outcome; Parkinson Disease; Pathway interactions; Physiological; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymers; Predisposition; Proteins; Regulation; Reperfusion Injury; Research; Resistance; Role; Site; Stimulus; Stroke; Therapeutic; Therapeutic Intervention; Tissues; Toxin; age related; cell type; excitotoxicity; in vivo; insight; mutant; prevent; tool

DESCRIPTION (provided by applicant): Significance of Caspase-Mediated Cleavage of PARP-1 in Cell Death Poly (ADP-ribose) polymerase-1 (PARP-1) maintains cell function by facilitating DNA repair. However, under pathological conditions, PARP-1 mediates cell death in experimental models of ischemia- reperfusion injury, myocardial infarction, glutamate excitotoxicity, and Parkinson disease. Thus, PARP-1 inhibition has been a therapeutic focus for aging-related diseases such as stroke, heart attack and neurodegeneration. PARP-1 facilitates DNA repair by producing poly(ADP-ribose) polymers (PAR) using NAD+ as a substrate, and attaching PAR to itself and other proteins. With excessive DNA damage however, PARP-1 overactivation leads to cell demise through depletion of NAD+ and overproduction of PAR. As such, understanding how PARP-1's activity is regulated during cell death will be valuable for halting aging and disease progression. One possible form of PARP-1 regulation during apoptotic cell death occurs when active caspases cleave PARP-1 into 89 and 24 kDa fragments possibly leading to PARP-1 inactivation. This cleavage has long been regarded as a biochemical hallmark of apoptosis, but how this cleavage affects PARP-1's physiological and pathological roles remains unclear. Since PARP-1's enzymatic function consumes NAD+, it is believed that this cleavage prevents PARP-1 over-activation from depleting cellular energy. Alternatively, in cell death pathways where PARP-1 promotes survival through DNA repair, PARP-1 cleavage may be necessary for the progression of cell death. To directly investigate the role of PARP- 1 cleavage in cell death, we generated a PARP-1 knock-in (PKI) mouse in which the caspase cleavage site of PARP-1 was mutated. We have confirmed that the mutation renders PARP-1 uncleavable by caspases in both neuronal and fibroblast cultures. How PARP-1 activity is affected by caspase cleavage will depend on the cell type studied and the death stimuli used. Thus, to delineate the cell death pathways in which caspase cleavage of PARP-1 are important, we will compare susceptibility of WT and PKI fibroblasts and neurons to a variety of cellular toxins. In addition, to determine the effect of PARP-1 cleavage in excitotoxic cell death, we will subject mice to experimental models of ischemic injury in which the brain injury is primarily mediated by PARP-1 over-activation. These studies will help to identify the functional significance of PARP-1 cleavage in different paradigms of cell death. With PARP-1's diverse roles in cellular homeostasis, aging, and disease, understanding how its activity is regulated may identify points of intervention for halting aging and disease progression. PARP-1 is cleaved by caspases during cell death, and this phenomenon has long been regarded as a biochemical hallmark for apoptosis. We believe that understanding the functional significance of this cleavage will help in developing therapeutic intervention to combat aging-related diseases and cell death.

描述（由申请方提供）：细胞死亡中Caspase介导的PARP-1切割的意义聚（ADP-核糖）聚合酶-1（PARP-1）通过促进DNA修复维持细胞功能。然而，在病理条件下，PARP-1在缺血-再灌注损伤、心肌梗死、谷氨酸兴奋性毒性和帕金森病的实验模型中介导细胞死亡。因此，PARP-1抑制已成为衰老相关疾病如中风、心脏病发作和神经变性的治疗焦点。PARP-1通过使用NAD+作为底物产生聚（ADP-核糖）聚合物（PAR）并将PAR附着于自身和其他蛋白质来促进DNA修复。然而，在过度DNA损伤的情况下，PARP-1过度活化通过NAD+的耗尽和PAR的过度产生导致细胞死亡。因此，了解PARP-1的活性在细胞死亡过程中是如何调节的，对于阻止衰老和疾病进展将是有价值的。当活性半胱天冬酶将PARP-1切割成89和24 kDa片段，可能导致PARP-1失活时，发生凋亡细胞死亡期间PARP-1调节的一种可能形式。这种切割一直被认为是细胞凋亡的生化标志，但这种切割如何影响PARP-1的生理和病理作用仍不清楚。由于PARP-1的酶功能消耗NAD+，因此认为这种切割防止PARP-1过度活化消耗细胞能量。或者，在PARP-1通过DNA修复促进存活的细胞死亡途径中，PARP-1裂解可能是细胞死亡进展所必需的。为了直接研究PARP- 1切割在细胞死亡中的作用，我们产生了PARP-1敲入（PKI）小鼠，其中PARP-1的半胱天冬酶切割位点发生突变。我们已经证实，突变使得PARP-1在神经元和成纤维细胞培养物中都不能被半胱天冬酶裂解。半胱天冬酶切割如何影响PARP-1活性将取决于所研究的细胞类型和所使用的死亡刺激。因此，为了描述其中PARP-1的半胱天冬酶裂解是重要的细胞死亡途径，我们将比较WT和PKI成纤维细胞和神经元对各种细胞毒素的易感性。此外，为了确定PARP-1裂解在兴奋性毒性细胞死亡中的作用，我们将小鼠置于缺血性损伤的实验模型中，其中脑损伤主要由PARP-1过度激活介导。这些研究将有助于确定PARP-1裂解在不同细胞死亡模式中的功能意义。由于PARP-1在细胞稳态、衰老和疾病中的不同作用，了解其活性如何调节可能会确定阻止衰老和疾病进展的干预点。PARP-1在细胞死亡过程中被半胱天冬酶切割，这种现象长期以来被认为是细胞凋亡的生化标志。我们相信，了解这种切割的功能意义将有助于开发治疗干预措施，以对抗衰老相关疾病和细胞死亡。