Dataset Information

CheA pupylation sites identified by SPIDER assay

ABSTRACT: We used CheAs and biotin-CheZ to validate the efficiency of SPIDER capturing protein-protein interaction. Pupylation sites on WT CheAs after SPIDER assay was identified by LC-MS/MS analysis, by searching for an additional mass of ~243 Da, which represents the three C-terminal residues of Pup, i. e., GGE, that covalently binds to adjacent lysine by pupylation. ）

ORGANISM(S): Escherichia Coli

SUBMITTER: Shengce Tao

PROVIDER: PXD026509 | iProX | Fri Jun 04 00:00:00 BST 2021

REPOSITORIES: iProX

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Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Jiang He-Wei HW Chen Hong H Zheng Yun-Xiao YX Wang Xue-Ning XN Meng Qingfeng Q Xie Jin J Zhang Jiong J Zhang ChangSheng C Xu Zhao-Wei ZW Chen Zi-Qing ZQ Wang Lei L Kong Wei-Sha WS Zhou Kuan K Ma Ming-Liang ML Zhang Hai-Nan HN Guo Shu-Juan SJ Xue Jun-Biao JB Hou Jing-Li JL Liu Zhe-Yi ZY Niu Wen-Xue WX Wang Fang-Jun FJ Wang Tao T Li Wei W Wang Rui-Na RN Dang Yong-Jun YJ Czajkowsky Daniel M DM Pei JianFeng J Dong Jia-Jia JJ Tao Sheng-Ce SC

Science China. Life sciences 20230414 8

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Repor ...[more]

PMID: 37059927

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Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value â¤ 0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 ÂµM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4Â°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.

Dataset Information

CheA pupylation sites identified by SPIDER assay

Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Similar Datasets

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