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.

NAD的ADP-核糖部分被转移到靶蛋白上，由细菌毒素和哺乳动物酶催化。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的症状。哺乳动物细胞含有酶，可催化类似于细菌毒素的反应。哺乳动物单ADP-核糖基转移酶（Arts）可以位于细胞和细胞表面，有时通过糖基磷脂酰肌醇（GPI）锚固链接（ART1）。其他Art5似乎是分泌的。几种哺乳动物单ADP-核糖基转移酶已克隆在实验室中。它们与毒素显示出一些结构相似性，在催化位点具有氨基酸的身份。转移酶催化的反应ADP-核糖 - （精氨酸）蛋白的产物被39 kDa ADP-核糖基金氨基氨基氨基水解酶（ARH1）裂解以再生未修饰的蛋白质。因此，转移酶和水解酶可以催化相反的反应构成ADP-核糖基化周期。 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。这些蛋白质参与了几个关键的生理过程，包括DNA修复，细胞分化和致癌过程。在人基因组中已经鉴定出多个聚（ADP-核糖）聚合酶，但是只有一种已知的聚（ADP-核糖）糖醇酶（PARG），这是一种111 kDA蛋白，它降低了（ADP-核糖）聚合物对ADP-核糖。小鼠和人类基因数据库中的另外两个蛋白质，39-kDa ARH2和ARH3似乎类似于ARH1。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）中产生ADP-核糖，但没有水解ADP-ribose-ribose-精氨酸，-ycysteine，-dipipiphthamide，-dipiphthamide of-Dipiphthamide和-asparagine bords。 39-KDA ARH3与ARH1和PARG的催化结构域具有氨基酸序列同一性。像ARH1一样，ARH3活性通过mg（2+）增强。鼠类ARH3的活性并不需要增强ARH1活性的硫醇。通过诱变（ASP（77）和ASP（78））鉴定的ARH3中的关键附属酸性氨基酸位于与ARH1（ASP（60）和ASP（61））活动所需的区域，但与PARG催化部位的关键生化型谷氨酸的位置不同。所有发现与ARH3具有PARG活性但结构与PARG无关的结论一致，除了催化域中的区域。 PARG家族的这个新成员可能与先前研究的酶具有不同的功能，并且可以在ADP-核糖代谢的调节中起特定的作用。 在人类气道和招募到气道的细胞中，上皮细胞部分通过释放人类嗜中性粒细胞肽（HNP）参与了先天免疫反应。我们先前报道说，这些细胞表面上精氨酸特异性的单-ADP-核糖基转移酶（ART）可以催化单核 - 核糖从NAD到蛋白质的转移。此外，我们注意到ART1是一种哺乳动物ADP-核糖基转移酶，存在于人类气道内的上皮细胞中，改性了HNP-1，从而改变了其功能。在来自哮喘，特发性肺纤维化或吸烟史的患者的支气管肺泡灌洗液（BALF）中鉴定出ADP-核糖基化的HNP-1（BALF）（活动中有两种常见的多态形式的ART1，在活动中有两种常见的多态形式），但在健康的志愿者或健康型淋巴结型或淋巴结型患者中却没有。在患有囊性纤维化的患者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这种修饰发生在气道中。实际上，来自具有吸烟史的个体的BALF中的大多数HNP-1都是单或DI-ADP-核糖基化的。 ART1在大肠杆菌中合成的Art1，糖基磷脂酰肌醇固醇恒温的ART1与磷脂酰肌醇特异性磷脂酶C释放出来的NMU细胞中的ART1或ART1在C2C12 Myotubes上在C2C12 Myotubes修饰14上的ART-1 with Artip-1 with Art-1 with Art-aftrination Artim-nney arnney arnn. arginine arnney arnney。大于ART3，ART4，ART5，铜绿假单胞菌外酶S或霍乱毒素A亚基的含量要大得多。小鼠ART2是NAD：精氨酸ADP-核糖基转移酶，能够修改HNP-1，但程度较小，而不是ART1。尽管ART5并未在很大程度上对HNP-1进行大量修改，但它抑制了ART5和ART1活动。人β-防御素-1（HBD1）是较差的转移酶底物。富含半胱氨酸的防御素的还原增强了它们作为ADP-核糖受体的能力。因此，分子的变性可能会暴露其他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，并且发生在炎症条件和疾病中。 从这些研究和其他研究中可以明显看出，NAD在细胞代​​谢的多个方面发挥作用，并通过酶通过酶传导，这些酶将ADP-核糖从NAD转移到受体蛋白上，从而改变其功能。 NAD是两种酶家族 - 单核-ADP-核糖基转移酶（MARTS）和聚（ADP-核糖）聚合酶（PARPS）的底物 - 共价转移ADP-核糖单体或聚合物，分别传递给受体蛋白。 ART2是MART，是在T淋巴细胞表面发现的免疫调节细胞的表型标记，包括肠内淋巴细胞（IELS）。我们已经表明，ART2.2等位基因蛋白的自动-ADP-核糖基化是多聚剂，涉及关键精氨酸（185）。我们的ART2和PARP的骨架结构比对表明，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。为了进行研究，我们使用了高度纯化的重组ART2，并证明ART2通过测序凝胶和HPLC和MS/MS/MS质谱法鉴定PR-AMP，这是PR-AMP，这是一种特定于聚（ADP-ribose）的分解产物。此外，我们将ART2上ADP-核糖聚合物附着的位点确定为R185，R185是其催化核心至关重要的环中的精氨酸。我们发现，IELS上的内源性ART2比外周T细胞上的ART2更有效地经历了多聚体自身ADP-核糖基化，这表明这些独特的淋巴细胞种群在ART2表面拓扑中有所不同。此外，与没有多聚体自动ADP-核糖基化活性的ART2.1 IEL相比，ART2.2 IEL对NAD诱导的细胞死亡具有更大的抵抗力。数据表明，聚合ADP-核糖的能力可能不是PARP所独有的，并且可以在细胞外和调节细胞功能的情况下进行聚（ADP-核糖化），一种已建立的核活性。