ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 7154203
• 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/7154203
• 关键词: ADP ribosylation; NAD nucleosidase; T lymphocyte; adenine phosphoribosyltransferase; bacterial toxins; enzyme activity; glycosylphosphatidylinositols; human subject; nicotinamide adenine dinucleotide; pentosyltransferase; posttranslational modifications; protein sequence; protein structure function; pyrophosphatase; tissue /cell culture

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 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 ADP-ribosyltransferases (ARTs) can be located within the cell and on the cell surface, sometimes linked through a glycosylphosphatidylinositol anchor (ART1). Others, ART5, appear to be secreted. A family of mammalian transferases has 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 (ADPRH)to regenerate unmodified protein. Thus, transferases and hydrolases can catalyze opposing reactions to constitute an ADP-ribosylation cycle. An ADPRH cDNA had been cloned from human, rat, and mouse tissues and high levels of hydrolase mRNA were found in brain, spleen, and testis. To begin to understand the molecular mechanisms that regulate ADPRH gene expression, we determined the genomic structure of mouse ADPRH, and investigated promoter function. Northern analyses using different regions of the ADPRH cDNA as probes identified mRNAs of 1.7 and 3.0 kb that resulted from the use of alternative polyadenylation signals, CATAAC and ATTAAA, beginning at positions 1501 and 2885, respectively, of the nucleotide sequence (A of ATG = 1). The ADPRH gene, represented in two overlapping genomic clones, spans 9 kb with four exons and three introns. The 5'-flanking region contains features of a housekeeping gene; it has neither a TATA nor a CAAT box, but is, instead, highly GC-rich with multiple transcription initiation sites. Promoter analysis, assessed using transient transfection of PC12, NB41A3, NIH/3T3, and Hepa 1-6 cells with truncated constructs, revealed potent stimulatory (-119 to -89) and inhibitory (-161 to -119) elements, which were utilized similarly in the different cell lines. Further mutational analysis of the promoter and electrophoretic mobility-shift assays identified a positive GC-box element (-107 to -95); Sp1 and Sp3, which bound to this motif, were also detected by supershift assays. In co-transfection experiments using Drosophila SL2 cells that lack endogenous Sp1, Sp1 trans-activated the ADPRH promoter in a manner dependent on the presence of an Sp1-binding motif. The promoter activity pattern and involvement of Sp transcription factors are consistent with prior observations of widespread hydrolase expression in mammalian tissues. The lungs of patients with cystic fibrosis (CF) are colonized frequently by Pseudomonas aeruginosa, which is associated with progressive lung destruction and increased mortality. A number of virulence factors, including exotoxin A (ETA) and the type III cytotoxins (ExoS, ExoT, ExoU, and ExoY) contribute to the pathogenicity of P. aeruginosa, which contacts the plasma membrane to deliver type III cytotoxins through a channel formed by many proteins, including PopB, PopD, and PcrV. ETA enters mammalian cells via receptor-mediated endocytosis. ETA, ExoS, and ExoT are ADP-ribosyltransferases that modify different substrates in mammalian cells. ETA, like diphtheria toxin, ADP-ribosylates elongation factor 2, thereby inhibiting protein synthesis. ExoS and ExoT target different signaling pathways, but they both ADP-ribosylate an arginine residue in their substrates. The recent study characterized the appearance with time of antibodies to components of the type III system in children with CF, who were colonized early in life with P. aeruginosa expressing the type III system. Surveillance for seroconversion to type III antigens in addition to clinical symptoms and oropharyngeal cultures may facilitate early detection of P. aeruginosa infections. Cholera toxin (CT), the toxic product produced by pathogenic agent responsible for cholera, exerts its effects on cells by ADP-ribosylation of a specific arginine in the regulatory guanine nucleotide-binding protein, G alpha As. Plant polyphenols, RG-tannin, and applephenon inhibited cholera toxin CT ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon, but not RG-tannin. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CT A subunit, and CT B subunit from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization. The natural products, or their derivatives may be useful in treating this toxin-mediated disease.

ADP-核糖基化NAD的ADP-核糖部分被转移到靶蛋白上，是由细菌毒素和哺乳动物酶家族催化的。一些毒素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ADP-核糖基转移酶（Arts）可以位于细胞内和细胞表面，有时通过糖基磷脂酰肌醇锚固链（ART1）链接。其他Art5似乎是分泌的。哺乳动物转移酶家族已被克隆在实验室中。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-核糖基氨基氨基氨基氨基水解酶（ADPRH）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。在大脑，脾脏和睾丸中发现了从人，大鼠和小鼠组织中克隆的ADPRH cDNA，并发现了高水平的水解酶mRNA。 为了开始了解调节ADPRH基因表达的分子机制，我们确定了小鼠ADPRH的基因组结构，并研究了启动子功能。使用ADPRH cDNA的不同区域作为探针进行的北部分析，该探针鉴定出是由使用替代聚腺苷酸化信号（CATAAC和ATTAAA）的1501和2885的位置，这是由替代聚腺苷酸化信号（a of ATG = a of ATG = a of ATG = a of ATG = a）。 1）。 ADPRH基因在两个重叠的基因组克隆中代表，跨越9 kb，带有四个外显子和三个内含子。 5'Fancing区域包含管家基因的特征。它既没有TATA也没有CAAT盒，而是具有多个转录启动位点的高度富含GC。启动子分析，使用PC12，NB41A3，NIH/3T3和HEPA 1-6细胞的瞬时转染具有截短构建体的细胞，揭示了有效的刺激性（-119至-89）和抑制（-161至-119），这些元素已被利用，这些元素已被利用同样在不同的细胞系中。对启动子和电泳迁移率迁移测定的进一步突变分析确定了阳性GC-box元件（-107至-107 -95）;与该基序结合的SP1和SP3也通过Supershift分析检测到。在使用缺乏内源性SP1的果蝇SL2细胞的共转染实验中，SP1以依赖于SP1结合基序的方式将ADPRH启动子转移。 SP转录因子的启动子活性模式和参与与先前观察到哺乳动物组织中广泛的水解酶表达。 囊性纤维化（CF）患者的肺部经常被铜绿假单胞菌定植，铜绿假单胞菌与进行性肺部破坏和死亡率增加有关。许多毒力因子，包括外毒素A（ETA）和III型细胞毒素（Exos，Exot，Exot，Exoy和Exoy）有助于铜绿假单胞菌的致病性，该致病性通过形成的III型细胞毒素通过形成的型质膜接触通过许多蛋白质，包括POPB，POPD和PCRV。 ETA通过受体介导的内吞作用进入哺乳动物细胞。 ETA，EXOS和EXOT是ADP-核糖基转移酶，可修饰哺乳动物细胞中不同底物。 ETA，像白喉毒素一样，ADP-核糖基延伸因子2，从而抑制蛋白质的合成。 EXOS和EXOT靶向不同的信号通路，但它们均ADP-核糖酸是其底物中的精氨酸残基。最近的研究表征了CF儿童III系统组件的抗体的外观，他们用铜绿假单胞菌表达了III型系统。除临床症状和口咽培养物外，还针对III型抗原的血清转化监测可能有助于早期发现铜绿假单胞菌感染。 霍乱毒素（CT）是导致霍乱的致病剂产生的毒性产物，通过在调节性鸟嘌呤核苷酸结合蛋白Gαg alpha AS中，特异性精氨酸的ADP-核糖基化对细胞发挥其对细胞的影响。植物多酚，RG-tannin和Applephenon抑制了霍乱毒素CT ADP核糖基转移酶活性和CT诱导的小鼠回肠环中的液体积累。高分子量的高分子量分数（HBT）也抑制了CT ADP-核糖基转移酶活性。 HBT和Applephenon以浓度依赖性的方式抑制CT与Vero细胞或GM1 GM1（CT受体）的结合，而不是RG-Tannin。随着毒素与细胞的结合，Applephenon，HBT和RG-Tannin抑制了其内在化。 HBT或Applephenon从溶液中沉淀了CT，CT A亚基和CT B亚基，从而产生大于250 kDa的聚集体。相反，RG - 单宁蛋白沉淀CT较差。它与CT，CTA或CTB形成了复合物，它们用蔗糖密度梯度离心和分子量排除过滤器证明。一致，CTA阻止了RG-Tannin对CT内在化的抑制。这些数据表明，某些类似于Applephenon和HBT的植物多酚结合CT，在溶液中形成大型聚集体，或者可能在细胞表面上形成大骨料，从而抑制CT结合和内在化。相反，RG单宁与CT的结合不会干扰其与Vero细胞或GM1的结合，但确实抑制了内在化。天然产物或其衍生物可能在治疗这种毒素介导的疾病中有用。