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Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

ABSTRACT: The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2-4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the ?-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.

SUBMITTER: Gan N

PROVIDER: S-EPMC6855250 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

Gan Ninghai N Zhen Xiangkai X Liu Yao Y Xu Xiaolong X He Chunlin C Qiu Jiazhang J Liu Yancheng Y Fujimoto Grant M GM Nakayasu Ernesto S ES Zhou Biao B Zhao Lan L Puvar Kedar K Das Chittaranjan C Ouyang Songying S Luo Zhao-Qing ZQ

Nature 20190722 7769

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system<sup>1</sup>. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery<sup>2-4</sup>. The activity of SidEs is regulated by another ...[more]

PMID: 31330531

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Project description:BackgroundHeterogeneous nuclear ribonucleoprotein K (HnRNPK) is a nucleic acid-binding protein that regulates diverse biological events. Pathologically, HnRNPK proteins are frequently overexpressed and clinically correlated with poor prognosis in various types of human cancers and are therefore pursued as attractive therapeutic targets for select patients. However, both the transcriptional regulation and degradation of HnRNPK in prostate cancer remain poorly understood.MethodsqRT-PCR was used to detect the expression of HnRNPK mRNA and miRNA; Immunoblots and immunohistochemical assays were used to determine the levels of HnRNPK and other proteins. Flow cytometry was used to investigate cell cycle stage. MTS and clonogenic assays were used to investigate cell proliferation. Immunoprecipitation was used to analyse the interaction between SPOP and HnRNPK. A prostate carcinoma xenograft mouse model was used to detect the in vivo effects of HnRNPK and miRNA.ResultsIn the present study, we noted that HnRNPK emerged as an important player in the carcinogenesis process of prostate cancer. miR-206 and miR-613 suppressed HnRNPK expression by targeting its 3'-UTR in PrCa cell lines in which HnRNPK is overexpressed. To explore the potential biological function, proliferation and colony formation of PrCa cells in vitro and tumor growth in vivo were also dramatically suppressed upon reintroduction of miR-206/miR-613. We have further provided evidence that Cullin 3 SPOP is a novel upstream E3 ubiquitin ligase complex that governs HnRNPK protein stability and oncogenic functions by promoting the degradation of HnRNPK in polyubiquitination-dependent proteolysis in the prostate cancer setting. Moreover, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of HnRNPK proteins.ConclusionOur findings reveal new posttranscriptional and posttranslational modification mechanisms of HnRNPK regulation via miR-206/miR-613 and SPOP, respectively. More importantly, given the critical oncogenic role of HnRNPK and the high frequency of SPOP mutations in prostate cancer, our results provide a molecular rationale for the clinical investigation of novel strategies to combat prostate cancer based on SPOP genetic status.

Dataset Information

Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

Publications

Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

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