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，其中NAD的ADP-核糖部分被转移到靶蛋白，由细菌毒素和哺乳动物酶家族催化。一些毒素ADP-核糖基转移酶（例如，霍乱毒素，白喉毒素）是导致由该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的反应的酶。哺乳动物ADP-核糖基转移酶（ART）可以位于细胞内和细胞表面上，有时通过糖基磷脂酰肌醇锚（ART 1）连接。其他的ART5似乎是分泌的。哺乳动物转移酶家族已在实验室中克隆;它们显示出与毒素的一些结构相似性，在催化位点具有氨基酸同一性。转移酶催化反应的产物ADP-核糖-（精氨酸）蛋白被39-kDa ADP-核糖基精氨酸水解酶（ADPRH）切割以再生未修饰的蛋白。因此，转移酶和水解酶可以催化相反的反应以构成ADP-核糖基化循环。已从人、大鼠和小鼠组织中克隆了ADPRH cDNA，并在脑、脾和睾丸中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的北方分析鉴定了1.7和3.0 kb的mRNA，这是由使用交替的多聚腺苷酸化信号CATAAC和ATTAAA产生的，分别从核苷酸序列的位置1501和2885开始（ATG的A = 1）。ADPRH基因，在两个重叠的基因组克隆，跨越9 kb的四个外显子和三个内含子。5'-侧翼区包含管家基因的特征;它既没有TATA也没有CAAT盒，而是具有多个转录起始位点的高度GC丰富。使用具有截短构建体的PC12、NB41A3、NIH/3T3和Hepa 1 - 6细胞的瞬时转染评估的启动子分析揭示了有效的刺激性（-119至-89）和抑制性（-161至-119）元件，其在不同细胞系中类似地使用。启动子的进一步突变分析和电泳迁移率变动分析鉴定了阳性GC盒元件（-107到108）。 -95）; Sp1和Sp3，结合到这个基序，也被supershift检测。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结合基序的存在的方式反式激活ADPRH启动子。启动子的活性模式和参与Sp转录因子是一致的广泛的水解酶在哺乳动物组织中表达的先前观察。 囊性纤维化（CF）患者的肺部经常被铜绿假单胞菌定植，这与进行性肺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、ExoU和ExoY）有助于铜绿假单胞菌的致病性，其接触质膜以通过由许多蛋白质（包括PopB、PopD和PcrV）形成的通道递送III型细胞毒素。ETA通过受体介导的内吞作用进入哺乳动物细胞。ETA、ExoS和ExoT是修饰哺乳动物细胞中不同底物的ADP-核糖基转移酶。ETA与白喉毒素一样，ADP-核糖基化延伸因子2，从而抑制蛋白质合成。ExoS和ExoT靶向不同的信号传导途径，但它们都使其底物中的精氨酸残基ADP-核糖基化。最近的研究表征了CF儿童中III型系统组分抗体随时间的出现，CF儿童在生命早期被表达III型系统的铜绿假单胞菌定殖。除了临床症状和口咽培养外，监测III型抗原的血清转化可能有助于铜绿假单胞菌感染的早期检测。 霍乱毒素（cholera toxin，CT）是由霍乱病原体产生的毒性产物，通过鸟嘌呤核苷酸结合蛋白G α As中的精氨酸的ADP-核糖基化作用对细胞产生影响。植物多酚，RG-单宁，和applephenon抑制霍乱毒素CT ADP-核糖基转移酶活性和CT诱导的液体积累在小鼠回肠袢。啤酒花苞叶提取物（HBT）的高分子量馏分也抑制CT ADP-核糖基转移酶活性。CT与Vero细胞或神经节苷脂GM1（CT受体）的结合以浓度依赖性方式被HBT和applephenon抑制，但不受RG-单宁的抑制。毒素结合细胞后，applephenon，HBT，和RG单宁抑制其内化。HBT或applephenon从溶液中沉淀CT、CT A亚基和CT B亚基，产生大于250 kDa的聚集体。与此相反，RG-单宁沉淀CT差，它形成复合物与CT，CTA，或CTB，这表明与蔗糖密度梯度离心和分子量排阻过滤器。与此一致，CTA阻断了RG-单宁对CT内化的抑制作用。这些数据表明，一些植物多酚，类似于applephenon和HBT，结合CT，在溶液中或细胞表面形成大的聚集体，从而抑制CT结合和内化。与此相反，RG单宁结合CT不干扰其结合Vero细胞或GM1，但它抑制内化。天然产物或其衍生物可用于治疗这种毒素介导的疾病。