ADP-ribosylation Cycles

解释 1.以前的研究表明，霍乱毒素是霍乱弧菌的产物，是一种以NAD为底物的ADP-核糖基转移酶，它催化ADP-核糖和烟酰胺的水解，以及ADP-核糖向蛋白质中游离精氨酸和精氨酸残基的转移。根据我们的发现，我们发现哺乳动物组织中存在NAD：精氨酸二磷酸核糖转移酶。然后我们问为什么ADP-核糖基化没有毒性，就像霍乱毒素的情况一样，并提出哺乳动物细胞有水解ADP-核糖-精氨酸(蛋白质)的酶。因此，精氨酸特异性的单-ADP-核糖化是一种可逆的翻译后修饰；精氨酸特异的霍乱毒素样单-ADP-核糖基转移酶(ARTC1s)将ADP-核糖从NAD+转移到精氨酸，然后ADP-核糖-精氨酸水解酶1(ARH1)裂解ADP-核糖-(精氨酸)蛋白键，生成未修饰的(精氨酸)蛋白。ARTC1已被证明可以增加肿瘤的致瘤性，而Arh1缺乏也会增加肿瘤的致瘤性。本研究建立了Artc1-KO和Artc1/Arh1-Double-KO小鼠，并对它们的特性进行了比较。Artc1基因缺陷小鼠的自发肿瘤形成减少，年龄相关的多器官炎症增加，并上调了促炎细胞因子肿瘤坏死因子(α)。在用致瘤的Arh1-KO小鼠胚胎成纤维细胞(MEF)建立的异种移植模型中，Artc1-KO和杂合受体小鼠的致瘤性降低，肿瘤被CD8+T细胞和巨噬细胞侵袭，导致坏死性下垂，这表明ARTc1促进了肿瘤微环境。此外，Artc1/Arh1-Double-KO MEF在裸鼠体内的成瘤率降低，因此肿瘤细胞和肿瘤微环境都需要ARTc1。通过超声心动图和核磁共振成像，Artc1-KO和杂合子小鼠显示出男性特有的心肌收缩能力降低。此外，与WT小鼠相比，Artc1-KO雄性心脏对心肌缺血-再灌注诱导的损伤具有更高的敏感性，受体相互作用蛋白激酶3(RIP3)蛋白水平增加，这表明Artc1抑制坏死性下垂。Artc1-KO的总体存活率低于其对应的Artc1-WT，主要是由于增强的免疫反应和炎症。因此，抗ARTC1药物可能会减少肿瘤的发生，但可能会增加多器官炎症和降低心脏收缩能力。 2.与COVID 19相关的研究：严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是导致全球600多万人死亡的全球大流行的罪魁祸首。该病毒编码几种非结构蛋白(NSP)，其中包含能够扰乱细胞过程的元素。在这些NSP蛋白中，NSP3含有大结构域，如MAC1、MAC2、MAC3，对宿主细胞具有潜在的作用。MAC1已被证明可以增加SARS-CoV-2的毒力，并破坏哺乳动物细胞中的ADP-核糖化途径。病毒大结构域已被证明可以裂解ADP-核糖-受体键，产生游离的ADP-核糖。通过这种反应，含有大结构域的蛋白质干扰宿主细胞中的ADP-核糖稳态。在这里，我们检测了SARS-CoV-2MAC1、2和3在含有ADP-核糖的底物上的潜在水解性。MAC1裂解-NAD+，但不裂解-NAD+，与C-1键的立体特异性一致。该反应类似于ARH1和ARH3以及其他大分子结构域所催化的反应。与ARH1和ARH3不同，MAC1的活性不需要镁离子。MAC1还能水解O-乙酰-ADP-核糖和ADP-核糖-1-磷酸，但MAC2和MAC3在这些反应中不起作用。此外，MAC1不能裂解-ADP-核糖-(精氨酸)和ADP-核糖-(丝氨酸)-组蛋白H3多肽，这表明MAC1对连接在O-和N-连接的官能团上的ADP-核糖具有特异性，其催化部位位于ADP-核糖部分。结论SARS-CoV-2 MAC1可能通过逆转宿主介导的ADP核糖化而发挥抗病毒作用。对NSP3大结构域活动的新见解可能有助于阐明SARS-CoV-2潜在的治疗靶点。

Explanation 1.Prior studies demonstrated that cholera toxin, the product of the bacteria Vibrio cholerae, is an ADP-ribosyltransferase that utilizes NAD as a substrate and catalyzes the hydrolysis to ADP-ribose and nicotinamide, as well as transfer of the ADP-ribose to free arginine and arginine residues in proteins. Others showed that the key modified protein was a guanine nucleotide-binding known as G(alpha)s. Based on our findings, we showed that mammalian tissues possessed NAD:arginine ADP-ribosyltransferases. We then asked why the ADP-ribosylation was not toxic, as is the case with cholera toxin, and proposed that mammalian cells had enzymes that hydrolyzed ADP-ribose-arginine(protein). Thus, arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTC1s) transfer ADP-ribose from NAD+ to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to increase tumorigenicity that is also increased by Arh1 deficiency. In this study, Artc1-KO and Artc1/Arh1-double-KO mice were generated and their properties compared. Artc1-deficient mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF(alpha). In a xenograft model using tumorigenic Arh1-KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1-KO and heterozygous recipient mice, with tumor infiltration by CD8+ T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1-double-KO MEFs showed decreased tumorigenesis in nude mice, thus both tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1-KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1-KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1-KO was less than their Artc1-WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility. 2.COVID 19-related studies: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. Viral macrodomains have been shown to cleave the ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere with ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD + , but not -NAD + , consistent with stereospecificity at the C-1 bond. This reaction is similar to those catalyzed by ARH1 and ARH3 as well as other macrodomains. In contrast to ARH1 and ARH3, Mac1 did not require Mg 2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1-phosphate, but Mac2 and Mac3 were inactive in these reactions. In addition, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 Macrodomain activities may shed light on potential SARS-CoV-2 therapeutic targets.