Ubiquitin-like Small Archaeal Protein Modification (SAMPylation)

泛素样小古菌蛋白修饰（SAMPylation）

• 批准号: 8231323
• 负责人: JULIE A MAUPIN-FURLOW
• 金额: $ 23.93万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231323
• 关键词: Affinity; Affinity Chromatography; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Archaea; Archaeal Proteins; Autophagocytosis; Biological Assay; C-terminal; Cell division; Cell physiology; Chemistry; Chromatography; Complex; DNA Repair; Development; Developmental Process; Disease; Enzymes; Epitopes; Escherichia coli; Eukaryota; Event; Florida; Fumarate Hydratase; Genes; Genetic Transcription; Glycine; Haloferax volcanii; Health; Homologous Gene; Human; Immunity; Immunoblotting; Immunoprecipitation; In Vitro; Knock-out; Link; Lysine; Malignant Neoplasms; Mediating; Mental Retardation; Metabolism; Metalloproteases; Methionine; Modeling; Modification; Monitor; Mono-S; Multienzyme Complexes; Mutation; N-terminal; Neurodegenerative Disorders; Nitrogen; Operon; Pathway interactions; Pharmaceutical Preparations; Phenotype; Play; Post-Translational Protein Processing; Process; Prokaryotic Cells; Proteins; Proteolysis; Recombinants; Recycling; Research; Role; Sampling; Signal Transduction; Site; Site-Directed Mutagenesis; Sulfur; System; Testing; Thiosulfate Sulfurtransferase; Ubiquitin; Ubiquitination; Universities; Variant; amino group; base; carboxyl group; design; enzyme activity; grasp; his6 tag; insight; interest; knockout gene; multicatalytic endopeptidase complex; mutant; protein degradation; protein transport; public health relevance; reconstitution; sulfoxide reductase; vector

DESCRIPTION (provided by applicant): Covalent attachment of selected cellular proteins to small protein modifiers of the 2-grasp fold ubiquitin/MoaD/ThiS superfamily plays an integral role in regulating a wide variety of metabolic and developmental processes related to human health. This includes the degradation of proteins by proteasomes, autophagy and non-proteolyitc events such as protein trafficking, DNA repair and cell signaling. While all eukaryotes encode ubiquitin- and ubiquitin-like-conjugating systems, the origin of these pathways is unclear and speculated to have assembled from various prokaryotic pathways that function in related, yet distinct, chemistry such as sulfur activation. This application is based on our recent discovery of two small archaeal protein modifiers (SAMP1 and SAMP2) that cluster to the 2-grasp fold superfamily, are highly conserved among archaea and are differentially conjugated to a large number of proteins in the halophilic archaeon Haloferax volcanii. The levels of SAMPylation were found to be modulated by environmental signals and proteasomal knockout mutations, thus providing early insight into how this modification may influence cell physiology. The chemistry of SAMPylation appears analogous in chemistry to ubiquitination based on the requirement of the C-terminal di-glycine motif of SAMP in covalent modification and the isolation of E1-, E2- and rhodanese-like SAMP conjugates. The aims of this project are to: 1) identify and classify the large group of Hfx. volcanii proteins that are conjugated to the SAMP1 and SAMP2 proteins (SAMPylated) and define the site and type of isopeptide bond formed in these modifications, 2) investigate whether the Hfx. volcanii proteins related to the E1-activating and E2-conjugating enzymes of ubiquitination that were isolated in complex with the SAMP proteins are required for SAMPylation, and 3) link the environmental signals and proteasomal gene knockouts that influence SAMP-conjugate formation to cell function. This application will test the following hypotheses: 1) SAMP1 and SAMP2 differentially conjugate a wide variety of proteins resulting in the formation of isopeptide bonds between the C-terminal carboxyl group of the SAMP protein and the 5-amino groups of the lysine residues of substrate proteins, 2) the E1 and E2 homologs of Hfx. volcanii function in the activation of SAMPs for transfer to the protein substrate and, 3) SAMPylation is likely to signal proteasome-mediated proteolysis in addition to non-proteolytic events. The proposed study of archaeal SAMPylation will provide new insight into 2- grasp Ub/MoaD/ThiS superfamily-mediated protein conjugation including an understanding of how this type of system functions in prokaryotic cells. PUBLIC HEALTH RELEVANCE: This research is related to ubiquitinylation and proteasome-mediated protein degradation, both of which are of great interest in human health. These pathways are emerging as key regulators of immunity, DNA repair and cell division, and their dysregulation is linked to a growing number of diseases including cancer, neurodegenerative disorders and mental retardation. Advancing our understanding of these processes will greatly assist in targeting proteasomes and Ub/Ubl-conjugation pathways for the development of new anti-cancer and anti-inflammatory drugs.

描述（由申请人提供）： 选定的细胞蛋白与2-rasp折叠泛素/MOAD/该超家族的共价附着在调节与人类健康相关的各种代谢和发育过程中起着不可或缺的作用。这包括蛋白酶体，自噬和非蛋白质溶液事件（例如蛋白质运输，DNA修复和细胞信号传导）降解蛋白质。尽管所有真核生物都编码泛素和泛素样结合系统，但这些途径的起源尚不清楚，并且推测是从各种核中途径中组装的，这些途径在相关但独特的化学作用（例如硫酸激活）中起作用。该应用是基于我们最近发现的两个小古细菌蛋白修饰剂（SAMP1和SAMP2）聚集到2-grasp折叠的超家族，在古细菌中是高度保守的，并且在卤素古老的haloferax volcanii中与大量蛋白质有差异共轭。发现Sampylation的水平是通过环境信号和蛋白酶体敲除突变调节的，从而提供了对这种修饰如何影响细胞生理的早期见解。基于SAMP的C末端二甘氨酸基序的要求，在化学中，Sampyration的化学在化学中似乎与泛素化相似，并在共价修饰中分离E1-，E2-和Rhodanese-like Samp samp conjugates。该项目的目的是：1）识别和对大型HFX进行分类。与SAMP1和SAMP2蛋白结合的火山蛋白（sampylated）并定义了这些修饰中形成的相位的位点和类型，2）研究是否研究了HFX。与Sampylation相关的泛素化酶与E1激活和E2偶联的酶有关的火山蛋白与SAMP蛋白需要分离，而Sampyl蛋白是需要的，3）将环境信号和蛋白酶体基因敲除影响SAMP结合形成samp偶联到细胞功能。该应用将检验以下假设：1）SAMP1和SAMP2差异结合了各种蛋白质，从而在SAMP蛋白的C端羧基与底物蛋白的赖氨酸蛋白的5-氨基群之间形成了异肽键。火山在激活剂量以转移到蛋白质底物的激活中，3）除非蛋白溶解事件外，sampylation可能还会信号蛋白酶体介导的蛋白水解。拟议的对古细胞化的研究将为2-抓取UB/MOAD/这项超家族介导的蛋白质结合提供新的见解，包括了解这种类型的系统在原核细胞中的功能。 公共卫生相关性： 这项研究与泛素化和蛋白酶体介导的蛋白质降解有关，这两种蛋白质都对人类健康引起了极大的兴趣。这些途径正在成为免疫，DNA修复和细胞分裂的关键调节剂，它们的失调与越来越多的疾病有关，包括癌症，神经退行性疾病和智力低下。促进我们对这些过程的理解将极大地有助于靶向蛋白酶体和UB/UBL结合途径，以开发新的抗癌和抗炎药。