ADP-ribosylation Cycles

ADP-核糖基化循环

• 批准号: 8158015
• 负责人: Joel Moss
• 金额: $ 147.92万
• 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/8158015
• 关键词: ADP ribosylation; ADP-Ribosylation Pathway; Active Sites; Adenosine Diphosphate Ribose; Affect; Agar; Alleles; Amino Acids; Animals; Arginine; Bacterial Toxins; Biological Assay; Chromatin Structure; Cleaved cell; Consensus; Cysteine; DNA; DNA Repair; Data; Development; Endoplasmic Reticulum; Enzymes; Exhibits; Exons; Family; Genes; Goals; Heterozygote; Histones; Hydrolase; Hydrolase Gene; Hydrolysis; Individual; Inositol; Integrin alpha1; Knockout Mice; Link; Longevity; Loss of Heterozygosity; Mammalian Cell; Modification; Mus; Mutate; Mutation; Niacinamide; Nucleotides; Nude Mice; Pathway interactions; Peptide Signal Sequences; Phospholipase C; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Post-Translational Protein Processing; Proteins; Reaction; Recombinant Proteins; Signal Pathway; Signal Transduction; Signaling Molecule; Smoking History; Subcutaneous Injections; Time; Tissues; Transferase; Tumor Tissue; Wild Type Mouse; cell transformation; human neutrophil peptide 1; prevent; protein function; tumorigenesis

In an earlier report, we showed that cells lacking the ARH1 gene showed enhanced proliferation compared to ARH1-/- cells transformed with a wild-type ARH1 gene; cells transformed with a mutant ARH1 gene (D60A/D61A), which encodes a protein that exhibits less than 0.1 percent of wild-type activity, also showed enhanced proliferation similar to ARH1-/- cells. ARH1-/- cells also produced colonies in soft agar, as did ARH1-/- cells transformed with the ARH1 gene containing the double mutation. In contrast, ARH1-/- cells transformed with the wild-type gene did not show colonies in soft agar. In further support for the tumorigenic potential of ARH1-/- cells, ARH1-/- cells and cells transformed with the mutated ARH1 gene also formed tumors in nude mice. In contrast, ARH1-/- cells transformed with the wild-type gene did not give rise to tumors when injected into nude mice. Of importance, ARH1+/- cells from heterozygous animals generated tumors in nude mice. In some heterozygous ARH1+/- mice, we also observed tumor development. We examined the functional allele from the tumors in nude mice injected with ARH1+/- cells or in tumors in heterozygous animals. In some instances, the functional allele appeared to be absent, consistent with loss of heterozygosity (LOH). These data are compatible with the autosomal dominant model of tumorigenesis proposed by Alfred Knudson, in which LOH occurs as the second hit leading to tumor formation. In other instances, however, there appeared to be an intact ARH1 allele in the heterozygote. The ARH1 gene from DNA isolated from those tumors was sequenced and contained nucleotide differences from the ARH1 gene in non-tumor DNA from nude mouse or in DNA from non-tumor tissue isolated from the heterozygote mouse. The mutant genes exhibited changes in exons 2 and 3, which are part of the coding region and may be responsible for the structure of the catalytic site and thus be expected to alter enzymatic activity. We next looked at the sequences of the mutant ARH1 gene from heterozygote cells injected into nude mice and from tumor obtained from heterozygote animals. To understand better the effects of the mutation, recombinant proteins were synthesized from the mutant genes and their activities were measured in a standard assay where the hydrolysis of ADP-ribose-arginine was quantified. In most cases, the mutant protein exhibited enzymatic activity, which was a fraction of the wild-type protein, consistent with the fact that the mutation occurred in a coding region exon, probably associated with the active site. When ARH1 KO cells were transfected with the mutant genes and the resulting transformed cells were injected subcutaneously into nude mice, tumors were observed in a time-dependent manner, even though the gene could be used to synthesize a protein exhibiting enzymatic activity. Thus, the presence of enzymatic activity is not sufficient to prevent tumor development. Conceivably, the mutation might affect the ability of the protein to be properly localized or to use different ADP-ribosylated proteins as substrates. The tumor growth rate following subcutaneous injection of KO cells was faster than those of cells transformed with a mutant gene, encoding an active ARH1. Thus, in quantitative assays, the presence or absence of a mutation may affect tumor development. KO cells transformed with a wild-type ARH1 also developed tumors depending on the level of cell lysate ARH1 activity. Cells having relatively low levels of ARH1 activity developed tumors in nude mice. Of note, when normalized to cell ARH1 enzymatic activitiy, transformation with the wild-type gene was more effective than transformation with mutant genes in slowing the rate of tumor development. Thus, abnormalities in the ARH1 gene appeared to determine, in part, the tumorigenic potential of the ARH1 KO cells.

在早期的报告中，我们表明，与野生型 ARH1 基因转化的 ARH1-/- 细胞相比，缺乏 ARH1 基因的细胞显示出更强的增殖能力；用突变型 ARH1 基因 (D60A/D61A) 转化的细胞（其编码的蛋白质的活性不到野生型活性的 0.1%）也显示出与 ARH1-/- 细胞类似的增殖增强。 ARH1-/- 细胞也在软琼脂中产生集落，用含有双突变的 ARH1 基因转化的 ARH1-/- 细胞也是如此。相反，用野生型基因转化的ARH1-/-细胞在软琼脂中没有显示集落。为了进一步支持ARH1-/-细胞的致瘤潜力，ARH1-/-细胞和用突变的ARH1基因转化的细胞也在裸鼠中形成肿瘤。相比之下，用野生型基因转化的ARH1-/-细胞注射到裸鼠体内时不会产生肿瘤。重要的是，来自杂合动物的 ARH1+/- 细胞在裸鼠中产生肿瘤。在一些杂合 ARH1+/- 小鼠中，我们还观察到肿瘤的发展。我们检查了注射 ARH1+/- 细胞的裸鼠肿瘤或杂合动物肿瘤的功能等位基因。在某些情况下，功能等位基因似乎不存在，这与杂合性丢失（LOH）一致。这些数据与 Alfred Knudson 提出的常染色体显性肿瘤发生模型相一致，其中 LOH 作为导致肿瘤形成的第二次打击而发生。然而，在其他情况下，杂合子中似乎存在完整的 ARH1 等位基因。对从这些肿瘤中分离的DNA中的ARH1基因进行测序，并发现其与裸鼠的非肿瘤DNA或从杂合子小鼠中分离的非肿瘤组织的DNA中的ARH1基因存在核苷酸差异。突变基因在外显子 2 和 3 中表现出变化，外显子 2 和 3 是编码区的一部分，可能负责催化位点的结构，因此预计会改变酶活性。 接下来，我们研究了注射到裸鼠体内的杂合子细胞和从杂合子动物获得的肿瘤中的突变 ARH1 基因的序列。为了更好地了解突变的影响，从突变基因合成了重组蛋白，并在标准测定中测量了它们的活性，其中对 ADP-核糖-精氨酸的水解进行了定量。在大多数情况下，突变蛋白表现出酶活性，这是野生型蛋白的一小部分，这与突变发生在编码区外显子中的事实一致，可能与活性位点相关。当用突变基因转染ARH1 KO细胞并将所得转化细胞皮下注射到裸鼠体内时，尽管该基因可用于合成具有酶活性的蛋白质，但仍以时间依赖性方式观察到肿瘤。因此，酶活性的存在不足以阻止肿瘤的发展。可以想象，突变可能会影响蛋白质正确定位或使用不同 ADP 核糖基化蛋白质作为底物的能力。皮下注射KO细胞后的肿瘤生长速度比用编码活性ARH1的突变基因转化的细胞更快。因此，在定量测定中，突变的存在或不存在可能会影响肿瘤的发展。用野生型 ARH1 转化的 KO 细胞也会产生肿瘤，具体取决于细胞裂解物 ARH1 活性的水平。 ARH1活性水平相对较低的细胞在裸鼠体内形成肿瘤。值得注意的是，当对细胞 ARH1 酶活性进行标准化时，野生型基因的转化比突变基因的转化在减慢肿瘤发展速度方面更有效。因此，ARH1 基因的异常似乎在一定程度上决定了 ARH1 KO 细胞的致瘤潜力。