{"doi":"10.21769/bioprotoc.3770","title":"Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ","abstract":"The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ-carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U-14C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This procedure is useful for the identification of substrates for glutamylation, characterization of substrate and glutamylase activities due to mutations, and identification of proteins with glutamylation activity. Some studies have assayed glutamylation with the use of [3H] glutamate (Regnard et al., 1998) and the use of the GT335 antibody (Wolff et al., 1992). However, the use of [U-14C] glutamate requires a shorter radioactive exposure time with no dependence on antibody specificity., [摘要]在军团菌效应蛋白SidJ最近被确定为另一个执行polyglutamylation军团效应，SdeA，烧蚀SdeA的活动。SidJ是一种激酶样蛋白，需要小的真核蛋白钙调蛋白才能进行谷氨酰化。谷氨酰化是翻译后修饰的相对不常见的类型，其中游离谷氨酸氨基酸的氨基与底物蛋白质中谷氨酸侧链的γ-羧基共价连接。该协议描述了使用放射性[U- 14]的SidJ谷氨酰化反应 C]谷氨酸及其底物SdeA，通过凝胶电泳分离蛋白质，制备用于放射暴露的凝胶，以及相对定量的谷氨酰化活性。该方法可用于鉴定底物进行谷氨酰化，表征底物和由突变引起的谷氨酰胺酶活性，以及​​鉴定具有谷氨酰化活性的蛋白质。一些研究已经使用[ 3 H]谷氨酸（Regnard等，1998）和使用GT335抗体（Wolff等，1992）来分析谷氨酰化。但是，使用[U- 14 C] g谷氨酸盐需要更短的放射性暴露时间，而不依赖于抗体特异性。[背景]嗜肺军团菌是感染细菌，造成军团病（麦克达德等人，1977） ，肺炎的一个潜在的致命的形式。在感染期间，Legionell a使用了300多种效应蛋白，其中许多蛋白具有不寻常的，无法识别的生化功能，可劫持宿主细胞的功能（Hubber和Roy，2010）。军团菌选择的一种方法是泛素化系统。研究表明，SidE蛋白家族可以对独立于E1和E2酶的底物蛋白进行磷酸核糖基泛素化（Bhogaraju等，2016； Qiu等，2016； Kotewicz等，2017）。一些研究暗示了SidJ在调节SidE蛋白质家族中的重要性，但尚未确定调节机制（Havey和Roy，2015； Jeong等，2015； Urbanas等，2016）。先前曾有人建议使用Legionella纯化的SidJ （Qiu和Luo，2019），将SidJ用作磷酸核糖基泛素化酶（Qiu等人，2017 ）; 然而，最近的研究并未复制这些结果（Bhogaraju等人，2019； Wan等人，2019； Shin等人，2020）。我们的团队（苏睿德等人，2019）和其他（Bhogaraju等，2019;黑色。等，2019;甘等人，2019） ，最近表明，SidJ可以polyglutamylate侧家人SdeA。为了验证这种活性，重要的是在体外反应中概括这些发现。SidJ具有C末端IQ螺旋，可以以钙非依赖性方式结合真核蛋白钙调蛋白。利用这种结合能力，通过X射线晶体学测定与钙调蛋白复合的SidJ的结构。SidJ包含一个激酶样结构域，与在激酶中发现的许多保守特征具有结构同源性。该激酶结构域通过与钙调蛋白的相互作用而处于活性构象。基于SidJ的这些特征，确定了反应成分并将其用于体外谷氨酰化测定。已经开发了使用放射性谷氨酸和通过液体闪烁的凝胶提取修饰的底物以检测修饰的其他测定法（Black等，2019）。液体闪烁和质谱可以分别提供对修饰的底物的量和附着在底物侧链上的谷氨酸盐数量的更精确定量。该协议中所述的测定方法可通过放射自显影和相对定量的活性显示活性。该测定法可用于鉴定SidJ底物并分析点突变对活性的影响。此外，该协议还可用于鉴定其他可充当谷氨酰胺酶的蛋白质或假激酶。","journal":"BIO-PROTOCOL","year":2020,"id":131168,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9586,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":456607,"name":"Jung‐Ho Shin","orcid":"0000-0001-9411-1331","position":1,"is_corresponding":false},{"id":585112,"name":"Marena Minelli","orcid":null,"position":2,"is_corresponding":false},{"id":456611,"name":"Yuxin Mao","orcid":"0000-0002-5064-1397","position":3,"is_corresponding":false},{"id":456608,"name":"Alan Sulpizio","orcid":"0000-0001-7112-8579","position":0,"is_corresponding":true}],"reference_count":19,"raw_metadata":null,"created_at":"2026-07-18T23:16:00.235845Z","pmid":"33659428","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}