ADP-ribosylation Cycles

ADP-核糖化是在一系列细菌毒素和哺乳动物酶的催化下，将NAD的ADP-核糖部分转移到靶蛋白上。一些毒素ADP-核糖基转移酶(如霍乱毒素、白喉毒素)导致由细菌引起的疾病的症状。哺乳动物细胞含有催化类似细菌毒素的反应的酶。哺乳动物ADP核糖基转移酶(ARTS)可定位于细胞内和细胞表面，有时通过糖基磷脂酰肌醇锚点连接(Art1)。其他的，ART5，似乎是秘密的。一个哺乳动物转移酶家族已经在实验室中被克隆，它们在结构上与毒素有一些相似之处，在催化部位有氨基酸的同一性。转移酶催化的产物ADP-核糖-精氨酸蛋白被39 kDa的ADP-核糖精氨酸水解酶(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-核糖化循环。已从人、大鼠和小鼠组织中克隆了ADPRH基因，并在脑、脾和睾丸中发现了高水平的水解酶mRNA。 为了开始了解调控ADPRH基因表达的分子机制，我们测定了小鼠ADPRH的基因组结构，并研究了启动子的功能。使用ADPRH基因不同区域作为探针的Northern分析发现，由于使用了交替的多聚腺苷信号CATAAC和ATTAAA，分别从核苷酸序列的1501和2885位开始(ATG=1的A)，mRNAs的大小分别为1.7和3.0kb。ADPRH基因存在于两个重叠的基因组克隆中，全长9kb，有4个外显子和3个内含子。5‘-侧翼区包含看家基因的特征；它既没有TATA，也没有CAAT盒，而是高度富含GC，具有多个转录起始点。用截短结构的PC12、NB41A3、NIH/3T3和HEPA 1-6细胞瞬时转染启动子分析，发现了有效的刺激(-119到-89)和抑制(-161到-119)元件，这些元件在不同的细胞系中得到了相似的利用。启动子的进一步突变分析和电泳迁移率改变分析确定了一个阳性的GC-box元件(-107到 -95)；超位移分析还检测到与该基序结合的Sp1和Sp3。在用缺乏内源性Sp1的果蝇SL2细胞进行的共转染实验中，Sp1以一种依赖于Sp1结合基序的方式反式激活ADPRH启动子。Sp转录因子的启动子活性模式和参与程度与先前观察到的哺乳动物组织中广泛表达的水解酶是一致的。 囊性纤维化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)参与了铜绿假单胞菌的致病性，它与质膜接触，通过包括POPB、PopD和PcrV在内的许多蛋白质形成的通道递送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是哺乳动物细胞中修饰不同底物的ADP核糖基转移酶。ETA和白喉毒素一样，ADP-核糖化延伸因子2，从而抑制蛋白质合成。EXOS和ExoT针对不同的信号通路，但它们都使底物中的精氨酸残基发生ADP核糖基化。最近的研究表征了针对III型系统成分的抗体在儿童CF中的出现随时间的变化，这些儿童在生命早期与表达III型系统的铜绿假单胞菌定居。除了临床症状和口咽培养外，监测血清转换为III型抗原可能有助于及早发现铜绿假单胞菌感染。 霍乱毒素(CT)是引起霍乱的病原体产生的有毒产物，它通过ADP核糖基化作用于鸟嘌呤核苷酸结合蛋白GαAS中的一种特定精氨酸而对细胞产生作用。植物多酚、RG-单宁和苹果酚可抑制霍乱毒素CTADP-核糖基转移酶活性和CT诱导的小鼠回肠积液。高相对分子质量的啤酒花苞叶提取物(HBT)也抑制CTADP-核糖基转移酶的活性。HBT和Apehenon以浓度依赖的方式抑制CT与Vero细胞或CT受体神经节苷脂GM1的结合，但不抑制RG-单宁。毒素与细胞结合后，苹果酚、HBT和RG-单宁抑制其内化。HBT或Apehenon使CT、CT A和CT B亚基从溶液中沉淀出来，形成大于250 kDa的聚集体。相反，RG-单宁对CT的沉淀效果较差；它与CT、CTA或CTB形成络合物，蔗糖密度梯度离心法和分子量排除滤膜证实了这一点。CTA可阻断RG-单宁对CT内化的抑制作用。这些数据表明，一些植物多酚，类似于苹果酚和HBT，与CT结合，在溶液中或在细胞表面形成大的聚集体，从而抑制CT的结合和内化。相反，RG-单宁与CT的结合不干扰其与Vero细胞或GM1的结合，但它确实抑制了内化。天然产物或其衍生物可能对治疗这种毒素介导的疾病有用。

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.