ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 7321530
• 负责人: Joel Moss
• 金额: --
• 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/7321530
• 关键词: ADP; ribosylation; Cycles

Mono-ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a target protein, is catalyzed by a family of bacterial toxins and mammalian enzymes. Some toxin mono-ADP-ribosyltransferases (e.g., cholera toxin, diphtheria toxin) are responsible for symptoms of the diseases caused by the bacterium. Mammalian cells contain enzymes that catalyze reactions similar to the bacterial toxins. Mammalian mono-ADP-ribosyltransferases (ARTs) can be located within the cell and on the cell surface, sometimes linked through a glycosylphosphatidylinositol (GPI) anchor (ART1). Others, ART5, appear to be secreted. Several of the mammalian mono-ADP-ribosyltransferases have been cloned in the laboratory; they display some structural similarities to the toxins, with amino acid identities in the catalytic site. A product of transferase-catalyzed reactions, ADP-ribose-(arginine)protein, is cleaved by a 39-kDa ADP-ribosylarginine hydrolase (ARH1)to regenerate unmodified protein. Thus, transferases and hydrolases can catalyze opposing reactions to constitute an ADP-ribosylation cycle. In addition to mono-ADP-ribosyltransferases, mammalian cells contain enzymes involved in poly(ADP-ribosylation); these proteins participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. Two other proteins in the mouse and human gene databases, the 39-kDa ARH2 and ARH3, appear to resemble ARH1. In the present study, we observed that the ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Thiols, which enhance the activity of ARH1 from some species, were not required for the activity of murine ARH3. Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 (Asp (60) and Asp (61)) but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG, except for regions in the catalytic domain. This new member of the PARG family might have different function(s) from the previously studied enzymes and could play a specific role(s) in the regulation of ADP-ribose metabolism. Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). We previously reported that arginine-specific mono-ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of mono-ADP-ribose from NAD to proteins. In addition, we noted that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in healthy volunteers or patients with lymphangioleiomyomatosis (LAM). Modified HNP-1 was not found in the sputum of patients with cystic fibrosis or in leukocyte granules of healthy volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. Denaturation of the molecule may thus expose additional ADP-ribose acceptor sites. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease. It is apparent from these and other studies that NAD functions in multiple aspects of cellular metabolism and signaling through enzymes that covalently transfer ADP-ribose from NAD to acceptor proteins, thereby altering their function. NAD is a substrate for two enzyme families-mono-ADP-ribosyltransferases (mARTs) and poly(ADP-ribose) polymerases (PARPs)-that covalently transfer an ADP-ribose monomer or polymer, respectively, to acceptor proteins. ART2, a mART, is a phenotypic marker of immunoregulatory cells found on the surface of T lymphocytes, including intestinal intraepithelial lymphocytes (IELs). We have shown that the auto-ADP-ribosylation of ART2.2 allelic protein is multimeric and involves a critical arginine (185). Our backbone structural alignment of ART2 and PARP suggested that multimeric auto-ADP-ribosylation of ART2 may represent an ADP-ribose polymer, rather than multiple sites of mono-ADP-ribosylation. To investigate this, we used highly purified recombinant ART2 and demonstrated that ART2 catalyzes the formation of an ADP-ribose polymer by sequencing gel and by HPLC and MS/MS mass spectrometry identification of PR-AMP, a breakdown product specific to poly(ADP-ribose). Further, we identified the site of ADP-ribose polymer attachment on ART2 as R185, an arginine in a crucial loop of its catalytic core. We found that endogenous ART2 on IELs undergoes multimeric auto-ADP-ribosylation more efficiently than ART2 on peripheral T cells, suggesting that these distinct lymphocyte populations differ in their ART2 surface topology. Furthermore, ART2.2 IELs are more resistant to NAD-induced cell death than ART2.1 IELs that do not have multimeric auto-ADP-ribosylation activity. The data suggest that capability of polymerizing ADP-ribose may not be unique to PARPs and that poly(ADP-ribosylation), an established nuclear activity, may occur extracellularly and modulate cell function.

单 ADP 核糖基化，即 NAD 的 ADP 核糖部分转移到靶蛋白上，由细菌毒素家族和哺乳动物酶催化。一些毒素单-ADP-核糖基转移酶（例如霍乱毒素、白喉毒素）是由细菌引起的疾病症状的原因。哺乳动物细胞含有催化类似于细菌毒素反应的酶。哺乳动物单 ADP 核糖基转移酶 (ART) 可以位于细胞内和细胞表面，有时通过糖基磷脂酰肌醇 (GPI) 锚 (ART1) 连接。其他的 ART5 似乎是分泌的。几种哺乳动物单 ADP 核糖基转移酶已在实验室中克隆；它们与毒素在结构上有一些相似之处，催化位点具有相同的氨基酸。转移酶催化反应的产物 ADP-核糖-（精氨酸）蛋白被 39-kDa ADP-核糖精氨酸水解酶 (ARH1) 裂解，重新生成未修饰的蛋白。因此，转移酶和水解酶可以催化相反的反应，构成ADP-核糖基化循环。 除了单-ADP-核糖基转移酶外，哺乳动物细胞还含有参与聚（ADP-核糖基化）的酶；这些蛋白质参与几个关键的生理过程，包括 DNA 修复、细胞分化和癌变。人类基因组中已鉴定出多种聚（ADP-核糖）聚合酶，但只有一种已知的聚（ADP-核糖）糖水解酶（PARG），它是一种 111 kDa 的蛋白质，可将（ADP-核糖）聚合物降解为 ADP-核糖。小鼠和人类基因数据库中的另外两种蛋白质，39-kDa ARH2 和 ARH3，似乎与 ARH1 相似。在本研究中，我们观察到 ARH1 样蛋白，称为聚（ADP-核糖）水解酶或 ARH3，表现出 PARG 活性，从聚（ADP-核糖）生成 ADP-核糖，但不水解 ADP-核糖-精氨酸、-半胱氨酸、-联苯胺或-天冬酰胺键。 39-kDa ARH3 与 ARH1 和 PARG 催化结构域具有相同的氨基酸序列。 ARH3 活性与 ARH1 一样，可被 Mg(2+) 增强。硫醇可以增强某些物种的 ARH1 活性，但小鼠 ARH3 的活性不需要硫醇。通过诱变鉴定的 ARH3 中的关键邻位酸性氨基酸（Asp(77) 和 Asp(78)）位于与 ARH1 活性所需区域相似的区域（Asp (60) 和 Asp (61)），但不同于PARG 催化位点中关键邻位谷氨酸的位置。所有发现都与 ARH3 具有 PARG 活性但在结构上与 PARG 无关（催化结构域中的区域除外）的结论一致。 PARG 家族的这个新成员可能具有与之前研究的酶不同的功能，并且可能在 ADP-核糖代谢的调节中发挥特定的作用。 人类气道内衬的上皮细胞和招募到气道的细胞部分通过释放人类中性粒细胞肽（HNP）来参与先天免疫反应。我们之前报道过，这些细胞表面的精氨酸特异性单 ADP 核糖基转移酶 (ART) 可以催化单 ADP 核糖从 NAD 转移到蛋白质。此外，我们注意到 ART1（一种哺乳动物 ADP-核糖基转移酶）存在于人类气道内壁上皮细胞中，它修饰了 HNP-1，改变了其功能。在患有哮喘、特发性肺纤维化或吸烟史（并且具有两种活性不同的 ART1 常见多态性）患者的支气管肺泡灌洗液 (BALF) 中发现了 ADP-核糖基化 HNP-1，但在健康志愿者或淋巴管平滑肌瘤病（LAM）患者。在囊性纤维化患者的痰液或健康志愿者的白细胞颗粒中未发现修饰的 HNP-1。在 BALF 中发现 ADP-核糖基-HNP-1，但在白细胞颗粒中没有发现，表明这种修饰发生在气道中。事实上，来自有吸烟史的个体的 BALF 中的大多数 HNP-1 都是单或双 ADP 核糖基化的。 ART1 在大肠杆菌中合成，糖基磷脂酰肌醇锚定的 ART1 与转染的 NMU 细胞中的磷脂酰肌醇特异性磷脂酶 C 一起释放，或在 C2C12 肌管上内源表达的 ART1 修饰了 HNP-1 上的精氨酸 14，并在精氨酸 24 上有二级位点。HNP- 的 ADP-核糖基化ART1 的 1 明显更高比 ART3、ART4、ART5、铜绿假单胞菌外切酶 S 或霍乱毒素 A 亚基产生的结果更有效。小鼠 ART2 是一种 NAD:精氨酸 ADP-核糖基转移酶，能够修饰 HNP-1，但程度低于 ART1。尽管HNP-1没有被ART5显着程度地修饰，但它抑制ART5以及ART1活性。人β-防御素-1 (HBD1) 是一种较差的转移酶底物。富含半胱氨酸的防御素的减少增强了它们作为 ADP-核糖受体的能力。因此，分子的变性可能会暴露额外的 ADP-核糖受体位点。我们得出的结论是，HNP-1 的 ADP-核糖基化似乎主要是 ART1 的活性，并且发生在炎症状况和疾病中。 从这些和其他研究中可以明显看出，NAD 通过将 ADP-核糖从 NAD 共价转移到受体蛋白的酶在细胞代谢和信号传导的多个方面发挥作用，从而改变其功能。 NAD 是两个酶家族（单 ADP 核糖基转移酶 (mART) 和聚 (ADP-核糖) 聚合酶 (PARP)）的底物，这两个酶家族分别将 ADP-核糖单体或聚合物共价转移至受体蛋白。 ART2（一种 mART）是 T 淋巴细胞（包括肠上皮内淋巴细胞 (IEL)）表面上发现的免疫调节细胞的表型标记。我们已经证明 ART2.2 等位蛋白的自身 ADP 核糖基化是多聚体，并且涉及关键的精氨酸 (185)。我们的 ART2 和 PARP 的主链结构比对表明 ART2 的多聚体自动 ADP-核糖基化可能代表 ADP-核糖聚合物，而不是单 ADP-核糖基化的多个位点。为了研究这一点，我们使用了高度纯化的重组 ART2，并通过测序凝胶以及 HPLC 和 MS/MS 质谱鉴定 PR-AMP（一种聚（ADP-核糖）特异性的分解产物）证明了 ART2 催化 ADP-核糖聚合物的形成。核糖）。此外，我们确定了 ART2 上 ADP-核糖聚合物附着位点为 R185，它是其催化核心关键环中的精氨酸。我们发现 IEL 上的内源性 ART2 比外周 T 细胞上的 ART2 更有效地进行多聚体自动 ADP 核糖基化，这表明这些不同的淋巴细胞群的 ART2 表面拓扑结构不同。此外，ART2.2 IEL 比不具有多聚体自动 ADP 核糖基化活性的 ART2.1 IEL 更能抵抗 NAD 诱导的细胞死亡。数据表明，聚合 ADP-核糖的能力可能并非 PARP 所独有，并且聚（ADP-核糖基化）（一种已确定的核活性）可能发生在细胞外并调节细胞功能。