Molecular Biology of the Ribonuclease A Gene Superfamily

核糖核酸酶 A 基因超家族的分子生物学

• 批准号: 7196724
• 负责人: HELENE ROSENBERG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/7196724
• 关键词: Molecular; Biology; Ribonuclease; Gene; Superfamily

During FY2005, we continued our exploration of the biology and evolution of the RNase A family of ribonucleases, an unusual enzyme family that is restricted to vertebrate species. While the chemistry of these enzymes has been carefully elucidated, the biological role of this highly divergent family remains for the most part unexplored. Our laboratory remains in the forefront of these biological studies, building on our past history which includes such highlights as the molecular cloning of human EDN/RNase 2 (PNAS 1989), human ECP/RNase 3 (J Exp Med 1989), elucidation of the unusual evolution of this lineage in primates (Nature Genetics 1995) and in rodents (PNAS 2000), structure function analysis of human EDN (PNAS 2002), identification and molecular cloning of human RNase 6 (NAR 1996), human RNase 8 (NAR 2002), and characterization of human EDN and ECP as antiviral ribonucleases (JID 1998; NAR 1998a, NAR 1998b). The first of our published manuscripts during FY2005 details the evolutionary divergence of mouse RNase 6. M. musculus RNase 6 has a limited expression pattern compared to human RNase 6 and is an efficient ribonuclease, with a catalytic efficiency 17-fold higher than that of human protein. Evolutionary analysis reveals that RNase 6 was subject to unusual evolutionary forces (dN/dS = 1.2) in an ancestral rodent lineage before the separation of Mus and Rattus. However, more recent evolution of rodent RNase 6 has been relatively conserved, with an average dN/dS of 0.66. These data suggest that the ancestral rodent RNase 6 was subject to accelerated evolution, resulting in the conserved modern gene, which most likely plays an important role in mouse physiology. The second manuscript described the first case of exon splicing among the members of the RNase A superfamily. Conserved among humans, mice and rats, the RNase 4 and RNase 5/ang 1 locus includes two non-coding exons followed by two distinct exons encoding RNase 4 and RNase 5/ang 1. Transcription from this locus is controlled by differential splicing and tissue-specific expression from promoters located 5' to each of the non-coding exons. Promoter 1, 5' to exon I, is universally active, while Promoter 2, 5' to exon II, is active only in hepatic cells in promoter assays in vitro. Transcription from Promoter 2 is dependent on an intact HNF-1 consensus binding site which binds the transcription factor HNF-1alpha. In summary, RNase 4 and RNase 5/ang 1 are unique among the RNase A ribonuclease genes in that they maintain a complex gene locus that is conserved across species with transcription initiated from tissue-specific dual promoters followed by differential exon splicing. The third manuscript describes the intronless open reading frame encoding an RNase A ribonuclease from genomic DNA from the Iguana iguana IgH2 cell line. The iguana RNase is expressed primarily in pancreas, and represents the majority of the specific enzymatic activity in this tissue. The encoded sequence shares many features with its better-known mammalian counterparts including the crucial His12, Lys40 and His114 catalytic residues and efficient hydrolytic activity against yeast tRNA substrate (k(cat)/K(m)=6 x 10(4) M(-1) s(-1)), albeit at a reduced pH optimum (pH 6.0). Although the catalytic activity of the iguana RNase is not diminished by human placental RI, iguana RNase is not bactericidal nor is it cytotoxic even at micromolar concentrations. Phylogenetic analysis indicates moderate (46%) amino acid sequence similarity to a pancreatic RNase isolated from Chelydra serpentina (snapping turtle) although no specific relationship could be determined between these RNases and the pancreatic ribonucleases characterized among mammalian species. Further analysis of ribonucleases from non-mammalian vertebrate species is needed in order to define relationships and lineages within the larger RNase A gene superfamily. We also collaborated with Dr. M. Victoria Nogues in order to assess the involvement of some cationic and aromatic surface exposed residues of ECP in the inhibition of proliferation of mammalian cell lines. We have constructed ECP mutants for the selected residues and assessed their ability to prevent cell growth. Trp10 and Trp35 together with the adjacent stacking residue are critical for the damaging effect of ECP on mammalian cell lines. These residues are also crucial for the membrane disruption activity of ECP. Other exposed aromatic residues packed against arginines (Arg75-Phe76 and Arg121-Tyr122) and specific cationic amino acids (Arg101 and Arg104) of ECP play a secondary role in the cell growth inhibition. This may be related to the ability of the protein to bind carbohydrates such as those found on the surface of mammalian cells.

在2005财政年度，我们继续探索核糖核酸酶的RNase A家族的生物学和进化，这是一种不寻常的酶家族，仅限于脊椎动物物种。虽然这些酶的化学性质已被仔细阐明，但这个高度分化的家族的生物学作用在很大程度上仍未被探索。我们的实验室仍然处于这些生物学研究的前沿，建立在我们过去的历史上，其中包括人类EDN/RNase 2的分子克隆等亮点（PNAS 1989），人ECP/RNase 3（J Exp Med 1989），阐明了灵长类动物中这一谱系的不寻常进化（Nature Genetics 1995）和啮齿类动物（PNAS 2000），人EDN的结构功能分析（PNAS 2002），人RNase 6的鉴定和分子克隆（NAR 1996）、人RNase 8（NAR 2002）和人EDN和ECP作为抗病毒核糖核酸酶的表征（JID 1998; NAR 1998 a，NAR 1998 b）。 我们在2005财年发表的第一篇论文详细描述了小鼠RNase 6的进化分歧。M.与人RNase 6相比，小家鼠RNase 6具有有限的表达模式，并且是一种有效的核糖核酸酶，其催化效率比人蛋白高17倍。进化分析表明，RNase 6受到不寻常的进化力量（dN/dS = 1.2）在祖先啮齿类动物谱系之前，小鼠和老鼠的分离。然而，啮齿动物RNase 6的最近进化相对保守，平均dN/dS为0.66。这些数据表明，祖先啮齿动物RNase 6经历了加速进化，导致了保守的现代基因，该基因很可能在小鼠生理学中起着重要作用。 第二份手稿描述了核糖核酸酶A超家族成员之间外显子剪接的第一例。RNase 4和RNase 5/ang 1基因座在人类、小鼠和大鼠中是保守的，包括两个非编码外显子，随后是编码RNase 4和RNase 5/ang 1的两个不同外显子。从该基因座的转录由位于每个非编码外显子5'的启动子的差异剪接和组织特异性表达控制。在体外启动子测定中，启动子1（外显子I的5'端）具有普遍活性，而启动子2（外显子II的5'端）仅在肝细胞中具有活性。启动子2的转录依赖于与转录因子HNF-1 α结合的完整HNF-1共有结合位点。总之，RNase 4和RNase 5/ang 1在RNase A核糖核酸酶基因中是独特的，因为它们保持了一个复杂的基因座，该基因座在物种间是保守的，转录起始于组织特异性双启动子，然后是差异外显子剪接。 第三份手稿描述了编码来自鬣蜥IgH 2细胞系基因组DNA的RNase A核糖核酸酶的无内含子开放阅读框。鬣蜥RNA酶主要在胰腺中表达，并且代表该组织中的大部分特异性酶活性。编码序列与其更知名的哺乳动物对应物共享许多特征，包括关键的His 12、Lys 40和His 114催化残基和对酵母tRNA底物的有效水解活性（k（cat）/K（m）=6 x 10（4）M（-1）s（-1）），尽管在降低的最佳pH值（pH 6.0）下。虽然鬣蜥RNA酶的催化活性不会被人胎盘RI减弱，但鬣蜥RNA酶即使在微摩尔浓度下也不具有杀菌性和细胞毒性。系统发育分析表明，中度（46%）的氨基酸序列相似性，从Chelydra serpentina（鳄龟）分离的胰腺核糖核酸酶，虽然没有特定的关系，可以确定这些核糖核酸酶和胰腺核糖核酸酶之间的哺乳动物物种的特点。需要对来自非哺乳类脊椎动物物种的核糖核酸酶进行进一步分析，以确定更大的核糖核酸酶A基因超家族内的关系和谱系。 我们也与M博士合作。维多利亚Nogues，以评估ECP的一些阳离子和芳香族表面暴露残基在抑制哺乳动物细胞系增殖中的参与。我们已经构建了所选残基的ECP突变体，并评估了它们阻止细胞生长的能力。Trp 10和Trp 35与相邻的堆积残基一起对于ECP对哺乳动物细胞系的损伤作用是至关重要的。这些残基对于ECP的膜破坏活性也至关重要。ECP的其他暴露的芳香残基（Arg 75-Phe 76和Arg 121-Tyr 122）和特定的阳离子氨基酸（Arg 101和Arg 104）在细胞生长抑制中起次要作用。这可能与蛋白质结合碳水化合物的能力有关，例如在哺乳动物细胞表面发现的碳水化合物。