ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 10008750
• 负责人: Joel Moss
• 金额: $ 214.21万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008750
• 关键词: 1-Phosphatidylinositol 3-Kinase; 8 year old; ADP Ribose Transferases; ADP ribosylation; Acute; Adenosine Diphosphate Ribose; Affect; Amino Acids; Animals; Annual Reports; Arginine; Attenuated; Bacterial Infections; Bacterial Toxins; Behavioral; Biosynthetic Proteins; Cardiac Myocytes; Cell Death; Cell Differentiation process; Cell Nucleus; Cell Survival; Cells; Cerebral Ischemia; Cessation of life; Cholera; Cholera Toxin; Cleaved cell; Clinical; Complex; Cyclic AMP; Cytoplasm; DNA; DNA Damage; DNA Repair; Data; Deacetylation; Diarrhea; Diphtheria; Disease; Electrolytes; Embryo; Enzymes; Epithelial Cells; Exhibits; Family; Family member; Female; Fibroblasts; Gender; Gender Sensitivity; Gene Mutation; Genes; Goals; Heart; High Pressure Liquid Chromatography; Human; Hydrogen Peroxide; Hydrolase; Hydrolysis; Injections; Injury; Intestines; Intoxication; Ischemia; Knock-out; Knockout Mice; Laboratory Study; Liquid substance; Lung; Mammalian Cell; Mediating; Membrane; Modification; Mus; Mutation; Myocardial; Myocardium; Nerve Degeneration; Neurologic; Neurons; Niacinamide; Nuclear; Nuclear Translocation; O-Acetyl-ADP-Ribose; Oxidative Stress; PHEMX gene; Pathogenesis; Pathway interactions; Patients; Pertussis Toxin; Phenotype; Phosphotransferases; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Post-Translational Protein Processing; Pre-Clinical Model; Protein Family; Proteins; Reaction; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporting; Role; Siblings; Signal Transduction; Sirtuins; Site; Skeletal Muscle; Stress; Symptoms; Tissues; Toxin; Vibrio cholerae; Virulence Factors; Wild Type Mouse; Woman; Wound Healing; apoptosis inducing factor; arm; base; biological systems; carcinogenesis; caveolin-3; cell injury; dimer; endonuclease; fast protein liquid chromatography; gender difference; genetic regulatory protein; inhibitor/antagonist; kidney epithelial cell; male; member; men; mouse model; poly ADP-ribose glycohydrolase; prevent; proband; progressive neurodegeneration; protein function; recruit; repaired; response

Explanation Mono-ADP-ribosylation is a post-translational protein modification, in which ADP-ribose is transferred from NAD to an acceptor amino acid. It was first identified as a mechanism of disease pathogenesis in the bacterial diseases such as cholera, diphtheria and pertussis, where toxins ADP-ribosylate critical regulatory and biosynthetic proteins. Mammalian tissues have enzymatic activities that mimic those of the bacterial toxins. Mammalian arginine-specific mono-ADP-ribosylation however, is a reversible modification of protein. Arginine-specific mono-ADP-ribosyltrans- ferases (ARTs) (e.g., ART1, ART5), transfer ADP-ribose from NAD to arginine residues of target proteins and ADP-ribosylarginine hydrolase 1 (ARH1) reverses the reaction by cleaving the ADP-ribose-(arginine)-protein bond. Data are consistent with ART and ARH1 serving as opposing arms of an arginine ADP-ribosylation cycle. 1. TRIM72 is an abundant in heart and skeletal muscle and in lung and kidney epithelial cells where it is involved in membrane repair. Effective membrane repair protects cardiomyocytes from ischemic damage. Oligomerization of TRIM72 was required acutely for membrane repair, bringing TRIM72 to the site of injury. TRIM72 also countered cell damage due to ischemia-reperfusion injury. TRIM72 in complex with caveolin-3 (Cav-3) activated phosphatidylinositol-3-kinases (PI3K)-dependent reperfusion injury salvage kinase (RISK), thereby enhancing cell survival. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in complexes, which were co-immunoprecipitated from mouse heart lysates. The complexes found in association with TRIM72 appeared to be relatively heterogeneous and could be resolved by FPLC and HPLC. Some of the complexes included Cav-3. It has been reported that TRIM72 oligomerization serves as a key mechanism for regulation of acute membrane repair. Oligomerization of TRIM72, observed in WT mice, was delayed in ARH1-deficient heart lysates, where a dimer of TRIM72 rather than a trimer was seen. In addition, at a cellular level, oligomerization of TRIM72 at the sites of injury required the presence of the mono-ADP-ribosylation cycle, that is, ART1 and ARH1. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury. 2. Enhanced sensitivity to cholera toxin in female ADP-ribosylarginine hydrolase (ARH1)-deficient mice. Cholera toxin, an 84-kDa multimeric protein and a major virulence factor of Vibrio cholerae, uses the ADP-ribosyltransferase activity of its A subunit to intoxicate host cells. ADP-ribosylation is a posttranslational modification of proteins, in which the ADP-ribose moiety of NAD+ is transferred to an acceptor. In mammalian cells, ADP-ribosylation of acceptors appears to be reversible. ADP-ribosyltransferases (ARTs) catalyze the modification of acceptor proteins, and ADP-ribose-acceptor hydrolases (ARHs) cleave the ADP-ribose-acceptor bond. ARH1 specifically cleaves the ADP-ribose-arginine bond. We previously demonstrated a role for endogenous ARH1 in regulating the extent of cholera toxin-mediated fluid and electrolyte abnormalities in a mouse model of intoxication. Murine ARH1-knockout (KO) cells and ARH1-KO mice exhibited increased sensitivity to cholera toxin compared to their wild-type (WT) counterparts. In the current report, we examined the sensitivity to cholera toxin of male and female ARH1-KO and WT mice. Intestinal loops derived from female ARH1-KO mice when injected with cholera toxin showed increased fluid accumulation compared to male ARH1-KO mice. WT mice did not show gender differences in fluid accumulation, ADP-ribosylarginine content, and ADP-ribosyl Gs levels. Injection of 8-Bromo-cAMP into the intestinal loops also increased fluid accumulation, however, there was no significant difference between female and male mice or in WT and KO mice. Female ARH1-KO mice showed greater amounts of ADP-ribosylated Gs protein and increased ADP-ribosylarginine content both in whole intestine and in epithelial cells than did male ARH1-KO mice. These results demonstrate that female ARH1-KO mice are more sensitive to cholera toxin than male mice. Loss of ARH1 confers gender sensitivity to the effects of cholera toxin but not of cyclic AMP. These observations may in part explain the finding noted in some clinical reports of enhanced symptoms of cholera and/or diarrhea in women than men. 3. PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells. Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient's deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient's fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency. 4. Collabotative studies are ongoing with Michael Hottiger and In-Kwon Kim.

解释 单 ADP 核糖基化是一种翻译后蛋白质修饰，其中 ADP 核糖从 NAD 转移至受体氨基酸。它首先被确定为霍乱、白喉和百日咳等细菌性疾病的疾病发病机制，其中毒素 ADP-核糖基化关键的调节和生物合成蛋白。哺乳动物组织具有模仿细菌毒素的酶活性。然而，哺乳动物精氨酸特异性单 ADP 核糖基化是蛋白质的可逆修饰。 精氨酸特异性单-ADP-核糖基转移酶 (ART)（例如 ART1、ART5）将 ADP-核糖从 NAD 转移到靶蛋白的精氨酸残基上，ADP-核糖精氨酸水解酶 1 (ARH1) 通过裂解 ADP-核糖-（精氨酸）-蛋白键来逆转该反应。数据与作为精氨酸 ADP-核糖基化循环的相反臂的 ART 和 ARH1 一致。 1. TRIM72 在心脏和骨骼肌以及肺和肾上皮细胞中含量丰富，参与膜修复。有效的膜修复可保护心肌细胞免受缺血性损伤。膜修复迫切需要 TRIM72 的寡聚化，从而将 TRIM72 带到损伤部位。 TRIM72 还可以对抗缺血再灌注损伤引起的细胞损伤。 TRIM72 与 Caveolin-3 (Cav-3) 复合物可激活磷脂酰肌醇 3 激酶 (PI3K) 依赖性再灌注损伤挽救激酶 (RISK)，从而增强细胞存活率。 调节酶 ART1 和 ARH1 及其底物 TRIM72 在复合物中被发现，该复合物是从小鼠心脏裂解物中进行免疫共沉淀的。发现的与 TRIM72 相关的复合物似乎相对异质，可以通过 FPLC 和 HPLC 解析。一些复合物包括 Cav-3。据报道，TRIM72 寡聚化是调节急性膜修复的关键机制。在 WT 小鼠中观察到的 TRIM72 寡聚化在 ARH1 缺陷的心脏裂解物中延迟，其中看到 TRIM72 二聚体而不是三聚体。 此外，在细胞水平上，TRIM72 在损伤部位的寡聚化需要单 ADP 核糖基化循环（即 ART1 和 ARH1）的存在。我们提出，涉及将 TRIM72 和其他调节因子募集到膜损伤位点的单 ADP-核糖基化循环对于心肌损伤后的膜修复和伤口愈合至关重要。 2. 雌性 ADP-核糖精氨酸水解酶 (ARH1) 缺陷小鼠对霍乱毒素的敏感性增强。 霍乱毒素是一种 84 kDa 多聚蛋白，是霍乱弧菌的主要毒力因子，利用其 A 亚基的 ADP-核糖基转移酶活性来毒害宿主细胞。 ADP-核糖基化是蛋白质的翻译后修饰，其中 NAD+ 的 ADP-核糖部分被转移到受体上。在哺乳动物细胞中，受体的 ADP-核糖基化似乎是可逆的。 ADP-核糖基转移酶 (ART) 催化受体蛋白的修饰，ADP-核糖受体水解酶 (ARH) 裂解 ADP-核糖-受体键。 ARH1 特异性裂解 ADP-核糖-精氨酸键。我们之前在小鼠中毒模型中证明了内源性 ARH1 在调节霍乱毒素介导的体液和电解质异常程度方面的作用。与野生型 (WT) 细胞相比，小鼠 ARH1 敲除 (KO) 细胞和 ARH1-KO 小鼠对霍乱毒素的敏感性更高。在本报告中，我们检查了雄性和雌性 ARH1-KO 和 WT 小鼠对霍乱毒素的敏感性。与雄性 ARH1-KO 小鼠相比，注射霍乱毒素后，雌性 ARH1-KO 小鼠的肠袢显示出液体积聚增加。 WT小鼠在体液积累、ADP-核糖精氨酸含量和ADP-核糖基Gs水平方面没有表现出性别差异。将 8-Bromo-cAMP 注射到肠袢中也会增加液体积聚，但雌性和雄性小鼠之间、WT 和 KO 小鼠之间没有显着差异。与雄性 ARH1-KO 小鼠相比，雌性 ARH1-KO 小鼠在全肠和上皮细胞中显示出更多的 ADP-核糖基化 Gs 蛋白和增加的 ADP-核糖精氨酸含量。这些结果表明雌性 ARH1-KO 小鼠比雄性小鼠对霍乱毒素更敏感。 ARH1 的缺失使性别对霍乱毒素的影响敏感，但对环 AMP 的影响不敏感。这些观察结果可能部分解释了一些临床报告中指出的女性霍乱和/或腹泻症状比男性更严重的发现。 3. PARP1 抑制可减轻 ARH3 缺陷小鼠和人类细胞的损伤。 聚（ADP-核糖基）化是指 ADP-核糖与蛋白质共价连接，产生支化的长链 ADP-核糖部分，称为聚（ADP-核糖）（PAR）。聚（ADP-核糖）聚合酶 1 (PARP1) 是响应 DNA 损伤的主要聚合酶和 PAR 受体。 PARP1 激活导致细胞内 PAR 过度积累，导致细胞死亡，途径称为“parthanatos”。 PAR 降解主要由聚 (ADP-核糖) 糖水解酶 (PARG) 和 ADP-核糖受体水解酶 3 (ARH3) 控制。我们之前的结果表明，ARH3 通过降低细胞质和细胞核 PAR 水平并防止细胞凋亡诱导因子 (AIF) 核转位，提供针对过氧化氢 (H2O2) 暴露的保护。我们发现了一个带有 ARH3 基因突变的家族，该突变导致蛋白质被截短、失活。这位8岁的先证者表现出进行性神经变性表型。此外，在患者已故兄弟姐妹的神经元中观察到了parthanatos，并且年长的兄弟姐妹表现出轻微的行为表型。与之前的研究结果一致，患者的成纤维细胞和 ARH3 缺陷小鼠分别对 H2O2 应激和脑缺血/再灌注诱导的 PAR 积累和细胞死亡更加敏感。此外，PARP1 抑制减轻了氧化应激和缺血/再灌注引起的细胞死亡和损伤。 PARP1 抑制剂可能会减轻 ARH3 缺陷患者神经退行性病变的进展。 4. 与 Michael Hottiger 和 In-Kwon Kim 正在进行合作研究。