Novel Insight on Nucleotide Regulation of Apoptosome Activation

关于凋亡体激活的核苷酸调控的新见解

• 批准号: 7795699
• 负责人: Dhyan Chandra
• 金额: $ 15.77万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7795699
• 关键词: Affect; Apoptosis; Apoptotic; Applications Grants; Binding; Biochemical; Bioenergetics; Caspase; Cell Death; Cells; Defect; Development; Event; Goals; High Pressure Liquid Chromatography; Imaging Techniques; Immunoprecipitation; In Vitro; Knowledge; Lead; Luciferases; Maintenance; Mammalian Cell; Mediating; Metabolism; Microinjections; Molecular; Nucleic Acids; Nucleotides; Physiological; Protein Isoforms; Proteins; Regulation; Resistance; Role; Stimulus; Testing; anti-cancer therapeutic; apoptotic protease-activating factor 1; caspase-3; caspase-9; cytochrome c; design; in vivo; insight; mutant; neoplastic; neoplastic cell; novel; prevent; reconstitution; stoichiometry; therapeutic target; tumor

DESCRIPTION (provided by applicant): Defects in apoptosis are intimately associated with tumor development and are also responsible for tumor cell resistance to anti-neoplastic agents. Caspase activation lies in the core of apoptotic cell death. Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in caspase-9 (an initiator caspase) activation, which ultimately activates effector caspases such as caspase-3 to execute cell demise. This cascade of caspase activation can be reconstituted in vitro with the addition of CC to cell lysates along with low levels of ATP or dATP (~10-200 ¿M), leading to the general conclusion by many that nucleotides such as dATP or ATP [(d)ATP] are proapoptotic. Most mammalian cells, however, possess an endogenous nucleotide pool of 4-10 mM. How the physiological, mM levels of ATP and nucleotides in general affect apoptosome formation and caspase activation remains a critical unanswered question. Recently, we have provided strong evidence that the physiologically relevant levels of nucleotides powerfully inhibit the CC-induced, apoptosome-mediated caspase-9 activation by binding directly to CC and preventing CC from interacting with Apaf-1. Consequently, the CC-mediated apoptosome assembly and activation are blocked. Co-microinjection of nucleotides and CC renders cells resistant to the CC-induced apoptosis in vivo whereas experimentally reducing nucleotides enhances both CC and apoptotic stimuli-induced cell death. These observations lead us to hypothesize that physiological levels of nucleotides, in addition to their well-established roles in nucleic acid synthesis, intermediate metabolism, and maintenance of bioenergetics, also function as critical prosurvival factors by directly inhibiting the CC-mediated apoptosome formation and caspase activation. In this K01 grant application, I propose three Specific Aims to test this hypothesis: 1) To further study nucleotide interaction with CC in vitro; 2) To elucidate nucleotide interaction with CC in vivo; and 3) To investigate nucleotide interaction with Apaf-1 and its impact on apoptosome activation. These aims will be accomplished using a combination of cellular, biochemical, and molecular approaches. The accomplishment of the proposed goals will significantly advance our understanding, at the molecular level, of how nucleotides regulate apoptosome and caspase activation as well as cell death. The newly obtained knowledge should also have important implications in helping to design novel anti-cancer therapeutics targeting the apoptotic machinery.

描述(由申请人提供)：细胞凋亡缺陷与肿瘤的发展密切相关，也是肿瘤细胞对抗肿瘤药物产生抗药性的原因。半胱氨酸天冬氨酸氨基转移酶激活是细胞凋亡的核心。细胞色素c(CC)启动的Apaf-1凋亡体的形成是caspase-9(一种启动子caspase)激活的关键启动事件，最终激活效应caspase，如caspase-3来执行细胞死亡。这种caspase激活的级联可以通过在细胞裂解产物中加入CC以及低水平的ATP或dATP(~10-200M)在体外重新构建，导致许多人普遍得出的结论是，dATP或ATP[(D)ATP]等核苷酸是促凋亡的。然而，大多数哺乳动物细胞拥有4-10 mm的内源核苷酸池。生理水平的三磷酸腺苷和核苷酸一般如何影响凋亡体的形成和半胱氨酸氨基转移酶的激活仍然是一个关键的未解答的问题。最近，我们提供了强有力的证据表明，生理相关水平的核苷酸通过直接与CC结合并阻止CC与APAF-1相互作用，有效地抑制了CC诱导的、凋亡体介导的caspase-9的激活。因此，CC介导的凋亡体组装和激活被阻断。在体内，联合微量注射核苷酸和CC使细胞对CC诱导的细胞凋亡产生抵抗，而实验中减少核苷酸会增强CC和凋亡刺激诱导的细胞死亡。 这些观察结果使我们推测，生理水平的核苷酸，除了在核酸合成、中间代谢和生物能量学的维持中已被证实的作用外，还通过直接抑制CC介导的凋亡体形成和caspase激活而发挥关键的生存因素的作用。在这项K01拨款申请中，我提出了三个具体的目的来验证这一假说：1)进一步研究核苷酸与CC的体外相互作用；2)阐明在体内与CC的核苷酸相互作用；3)研究核苷酸与APAF-1的相互作用及其对凋亡体激活的影响。这些目标将使用细胞、生物化学和分子方法的组合来实现。这些目标的实现将大大提高我们在分子水平上对核苷酸如何调节凋亡体和caspase激活以及细胞死亡的理解。这些新获得的知识也应该对帮助设计针对细胞凋亡机制的新型抗癌疗法具有重要意义。

项目成果

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation.

• DOI: 10.1016/j.mito.2012.10.010
• 发表时间: 2013-09
• 期刊: MITOCHONDRION
• 影响因子: 4.4
• 作者: Prabhu, Varun;Srivastava, Pragya;Yadav, Neelu;Amadori, Michael;Schneider, Andrea;Seshadri, Athul;Pitarresi, Jason;Scott, Rachael;Zhang, Honghao;Koochekpour, Shahriar;Gogada, Raghu;Chandra, Dhyan
• 通讯作者: Chandra, Dhyan

Neem oil limonoids induces p53-independent apoptosis and autophagy.

• DOI: 10.1093/carcin/bgs269
• 发表时间: 2012-11
• 期刊: Carcinogenesis
• 影响因子: 4.7
• 作者: P. Srivastava;Neelu Yadav;Ravi K. Lella;Andrea Schneider;Anthony Jones;T. Marlowe;Gabrielle Lovett

Mitochondrial DNA mutations and breast tumorigenesis.

• DOI: 10.1016/j.bbcan.2013.10.002
• 发表时间: 2013-12
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER
• 影响因子: 11.2
• 作者: Yadav, Neelu;Chandra, Dhyan
• 通讯作者: Chandra, Dhyan

Molecular insights on cytochrome c and nucleotide regulation of apoptosome function and its implication in cancer.

• DOI: 10.1016/j.bbamcr.2019.118573
• 发表时间: 2020-01
• 期刊: Biochimica et biophysica acta. Molecular cell research
• 作者: Yadav N;Gogada R;O'Malley J;Gundampati RK;Jayanthi S;Hashmi S;Lella R;Zhang D;Wang J;Kumar R;Suresh Kumar TK;Chandra D
• 通讯作者: Chandra D