ADP-ribosylation Cycles

解释 1.先前的研究表明，霍乱毒素是细菌霍乱弧菌的产物，是一种ADP-核糖基转移酶，其利用NAD作为底物并催化水解为ADP-核糖和烟酰胺，以及将ADP-核糖转移为游离精氨酸和蛋白质中的精氨酸残基。其他研究表明，关键的修饰蛋白质是一种称为G（alpha）的鸟嘌呤核苷酸结合蛋白。基于我们的研究结果，我们表明，哺乳动物组织具有NAD：精氨酸ADP-核糖基转移酶。然后我们询问为什么ADP-核糖基化没有毒性，就像霍乱毒素一样，并提出哺乳动物细胞具有水解ADP-核糖-精氨酸（蛋白质）的酶。因此，精氨酸特异性单ADP核糖基化是一种可逆的翻译后修饰;精氨酸特异性霍乱毒素样单ADP核糖基转移酶（ARTC 1）将ADP-核糖从NAD+转移至精氨酸，然后通过ADP-核糖基精氨酸水解酶1（ARH 1）切割ADP-核糖-（精氨酸）蛋白键，产生未修饰的（精氨酸）蛋白。 ARTC 1已显示出增加致瘤性，Arh 1缺乏也会增加致瘤性。在这项研究中，Artc 1-KO和Artc 1/Arh 1-double-KO小鼠的产生和它们的性能进行了比较。Artc 1缺陷小鼠表现出自发性肿瘤发生减少，年龄依赖性多器官炎症增加，促炎细胞因子TNF（α）上调。在使用致瘤性Arh 1-KO小鼠胚胎成纤维细胞（MEF）的异种移植模型中，Artc 1-KO和杂合受体小鼠的致瘤性降低，CD 8 + T细胞和巨噬细胞浸润肿瘤，导致坏死性凋亡，表明ARTC 1促进肿瘤微环境。此外，Artc 1/Arh 1-双KO MEFs在裸鼠中显示出肿瘤发生减少，因此肿瘤细胞以及肿瘤微环境都需要ARTC 1。通过超声心动图和MRI，Artc 1-KO和杂合子小鼠表现出雄性特异性心肌收缩力降低。此外，与WT小鼠相比，Artc 1-KO雄性心脏表现出对心肌缺血-再灌注诱导的损伤的敏感性增强，受体相互作用蛋白激酶3（RIP 3）蛋白水平增加，表明ARTC 1抑制坏死性凋亡。Artc 1-KO的总体存活率低于Artc 1-WT，主要是由于增强的免疫应答和炎症。因此，抗ARTC 1药物可能减少肿瘤发生，但可能增加多器官炎症和降低心脏收缩力。 2. COVID 19相关研究：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病的原因。该病毒编码几种非结构蛋白（Nsps），其中含有能够破坏细胞过程的元件。在这些Nsp蛋白中，Nsp 3含有宏结构域，例如，Mac 1、Mac 2、Mac 3，对宿主细胞具有潜在作用。Mac 1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-核糖基化途径。已显示病毒大结构域切割ADP-核糖-受体键，产生游离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，我们研究了SARS-CoV-2 Mac 1，2和3对含有ADP-核糖的底物的潜在水解活性。Mac 1切割-NAD +，但不切割-NAD +，与C-1键的立体特异性一致。该反应类似于由ARH 1和ARH 3以及其他宏域催化的那些反应。与ARH 1和ARH 3不同，Mac 1不需要Mg 2+来维持最佳活性. Mac 1还水解O-乙酰基-ADP-核糖和ADP-核糖-1-磷酸，但Mac 2和Mac 3在这些反应中是无活性的。此外，Mac 1不切割ADP-核糖-（精氨酸）和ADP-核糖-（丝氨酸）-组蛋白H3肽，表明Mac 1水解连接到O-和N-连接的官能团的ADP-核糖，在ADP-核糖部分的催化位点具有特异性。我们的结论是SARS-CoV-2 Mac 1可能通过逆转宿主介导的ADP-核糖基化来发挥抗病毒活性。对Nsp 3宏结构域活性的新见解可能揭示潜在的SARS-CoV-2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.