Ubiquitin-like Small Archaeal Protein Modification (SAMPylation)

泛素样小古菌蛋白修饰（SAMPylation）

• 批准号: 8438503
• 负责人: JULIE A MAUPIN-FURLOW
• 金额: $ 23.09万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8438503
• 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; 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; 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.

描述（由申请人提供）： 选定的细胞蛋白与 2-grasp Fold ubiquitin/MoaD/ThiS 超家族的小蛋白修饰剂的共价连接在调节与人类健康相关的各种代谢和发育过程中发挥着不可或缺的作用。这包括蛋白酶体、自噬和非蛋白水解事件（例如蛋白质运输、DNA 修复和细胞信号传导）对蛋白质的降解。虽然所有真核生物都编码泛素和类泛素结合系统，但这些途径的起源尚不清楚，推测是由各种原核生物途径组装而成，这些原核生物途径在相关但独特的化学（如硫活化）中发挥作用。该应用基于我们最近发现的两种小型古细菌蛋白修饰剂（SAMP1 和 SAMP2），它们属于 2-grasp Fold 超家族，在古细菌中高度保守，并且与嗜盐古细菌 Haloferax volcanii 中的大量蛋白质有差异地缀合。人们发现 SAMPylation 的水平受到环境信号和蛋白酶体敲除突变的调节，从而为了解这种修饰如何影响细胞生理学提供了早期见解。基于共价修饰中 SAMP 的 C 端二甘氨酸基序的要求以及 E1-、E2- 和硫氰酸酶样 SAMP 缀合物的分离，SAMP 化的化学性质在化学上与泛素化类似。该项目的目标是：1）对 Hfx 大群体进行识别和分类。 volcanii 蛋白与 SAMP1 和 SAMP2 蛋白（SAMPylated）缀合，并定义这些修饰中形成的异肽键的位点和类型，2) 研究 Hfx 是否。与泛素化 E1 激活酶和 E2 结合酶相关的火山蛋白与 SAMP 蛋白复合分离，是 SAMP 化所必需的，3) 将影响 SAMP 结合物形成的环境信号和蛋白酶体基因敲除与细胞功能联系起来。该应用将测试以下假设：1) SAMP1 和 SAMP2 差异性地缀合多种蛋白质，导致 SAMP 蛋白质的 C 端羧基与底物赖氨酸残基的 5-氨基之间形成异肽键蛋白质，2) Hfx 的 E1 和 E2 同系物。 volcanii 在激活 SAMP 以转移至蛋白质底物中发挥作用，并且，3) 除非蛋白水解事件外，SAMP 化可能还发出蛋白酶体介导的蛋白水解作用的信号。拟议的古菌 SAMPylation 研究将为 2-抓握 Ub/MoaD/ThiS 超家族介导的蛋白质缀合提供新的见解，包括了解这种类型的系统如何在原核细胞中发挥作用。