Dataset Information

Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB.

ABSTRACT: Legionella pneumophila is an intracellular pathogen that requires nutrients from the host for its replication. It has been shown that replicating L. pneumophila prefers amino acids as main sources of carbon and energy. The homeostasis of amino acids in eukaryotic cells is regulated by the transcription factor EB (TFEB), which translocates into the nucleus and activates genes for autophagy and lysosomal biogenesis. Here we show that the Legionella effector SetA causes a robust nuclear translocation of TFEB when exogenously expressed in mammalian cells and that the translocation is dependent on the glucosyltransferase activity of SetA. We further show that SetA directly glucosylates TFEB at multiple sites. Our findings of TFEB glucosylation by SetA may suggest an alternative strategy for exploiting host nutrients in addition to the control of host mTORC1 signaling by L. pneumophila. Our results provide further insight into the molecular mechanism of the delicate TFEB nuclear shuttling.

SUBMITTER: Beck WHJ

PROVIDER: S-EPMC7334434 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB.

Beck Wendy H J WHJ Kim Dongsung D Das Jishnu J Yu Haiyuan H Smolka Marcus B MB Mao Yuxin Y

iScience 20200620 7

Legionella pneumophila is an intracellular pathogen that requires nutrients from the host for its replication. It has been shown that replicating L. pneumophila prefers amino acids as main sources of carbon and energy. The homeostasis of amino acids in eukaryotic cells is regulated by the transcription factor EB (TFEB), which translocates into the nucleus and activates genes for autophagy and lysosomal biogenesis. Here we show that the Legionella effector SetA causes a robust nuclear translocati ...[more]

PMID: 32622269

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Project description:BACKGROUND:The intracellular bacterial pathogen Legionella pneumophila proliferates in human alveolar macrophages, resulting in a severe pneumonia termed Legionnaires' disease. Throughout the course of infection, L. pneumophila remains enclosed in a specialized membrane compartment that evades fusion with lysosomes. The pathogen delivers over 300 effector proteins into the host cell, altering host pathways in a manner that sets the stage for efficient pathogen replication. The L. pneumophila effector protein AnkX targets host Rab GTPases and functions in preventing fusion of the Legionella-containing vacuole with lysosomes. However, the current understanding of AnkX's interaction with host proteins and the means through which it exerts its cellular function is limited. RESULTS:Here, we investigated the protein interaction network of AnkX by using the nucleic acid programmable protein array (NAPPA), a high-density platform comprising 10,000 unique human ORFs. This approach facilitated the discovery of PLEKHN1 as a novel interaction partner of AnkX. We confirmed this interaction through multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Structured illumination microscopy revealed that endogenous PLEKHN1 is found in the nucleus and on vesicular compartments, whereas ectopically produced AnkX co-localized with lipid rafts at the plasma membrane. In mammalian cells, HaloTag-AnkX co-localized with endogenous PLEKHN1 on vesicular compartments. A central fragment of AnkX (amino acids 491-809), containing eight ankyrin repeats, extensively co-localized with endogenous PLEKHN1, indicating that this region may harbor a new function. Further, we found that PLEKHN1 associated with multiple proteins involved in the inflammatory response. CONCLUSIONS:Altogether, our study provides evidence that in addition to Rab GTPases, the L. pneumophila effector AnkX targets nuclear host proteins and suggests that AnkX may have novel functions related to manipulating the inflammatory response.

Project description:RATIONALE:Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. OBJECTIVE:We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. METHODS AND RESULTS:In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-? signaling and upregulated autophagy. Through inactivation of AMPK? or inhibition of autophagy, we demonstrated that the AMPK? and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPK? activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). CONCLUSIONS:In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPK? and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Project description:Vascular smooth muscle cells (VSMCs) contribute to plaque stability. VSMCs are also a major source of CTH (cystathionine gamma-lyase)-hydrogen sulfide (H2S), a protective gasotransmitter in atherosclerosis. However, the role of VSMC endogenous CTH-H2S in pathogenesis of plaque stability and the mechanism are unknown. In human carotid plaques, CTH expression in ACTA2+ cells was dramatically downregulated in lesion areas in comparison to non-lesion areas. Intraplaque CTH expression was positively correlated with collagen content, whereas there was a negative correlation with CD68+ and necrotic core area, resulting in a rigorous correlation with vulnerability index (r = -0.9033). Deletion of Cth in VSMCs exacerbated plaque vulnerability, and were associated with VSMC autophagy decline, all of which were rescued by H2S donor. In ox-LDL treated VSMCs, cth deletion reduced collagen and heightened apoptosis association with autophagy reduction, and vice versa. For the mechanism, CTH-H2S mediated VSMC autophagosome formation, autolysosome formation and lysosome function, in part by activation of TFEB, a master regulator for autophagy. Interference with TFEB blocked CTH-H2S effects on VSMCs collagen and apoptosis. Next, we demonstrated that CTH-H2S sulfhydrated TFEB at Cys212 site, facilitating its nuclear translocation, and then promoting transcription of its target genes such as ATG9A, LAPTM5 or LDLRAP1. Conclusively, CTH-H2S increases VSMC autophagy by sulfhydration and activation of TFEB, promotes collagen secretion and inhibits apoptosis, thereby attenuating atherogenesis and plaque vulnerability. CTH-H2S may act as a warning biomarker for vulnerable plaque.Abbreviations ATG9A: autophagy related 9A; CTH: cystathionine gamma-lyase; CQ: chloroquine; HASMCs: human aortic smooth muscle cells; H2S: hydrogen sulfide; LAMP1: lysosomal associated membrane protein 1; LAPTM5: lysosomal protein transmembrane 5; NaHS: sodium hydrosulfide hydrate; ox-LDL: oxidized-low density lipoprotein; PPG: DL- propagylglycine; TFEB: transcription factor EB; 3-MA: 3-methyladenine; VSMCs: vascular smooth muscle cells.

Dataset Information

Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB.

Publications

Glucosylation by the Legionella Effector SetA Promotes the Nuclear Localization of the Transcription Factor TFEB.

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