Dataset Information

CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1.

ABSTRACT: Cereblon (CRBN) as a multifunctional protein has been extensively studied. Here, we show that CRBN is a negative regulator of bactericidal activity and autophagy activation. Mitochondrial localization of CRBN was significantly increased in response to Toll-like receptor 4 (TLR4) stimulation. CRBN interrupted the association of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT)-TNF-receptor associated factor 6 (TRAF6) complex, thereby inhibiting the ubiquitination of ECSIT, which plays a pivotal role for the production of mitochondrial reactive oxygen species (mROS). Subsequently, mROS levels were markedly elevated in CRBN-knockdown (CRBNKD) THP-1 cells, and that led to resistance against S. typhimurium infection, indicating CRBN is a negative regulator of bactericidal activity through the regulation of mROS. Additionally, CRBN inhibited TRAF6-induced ubiquitination of BECN1 (Beclin 1), and that induced autophagy activation in CRBNKD THP-1, CRBN-knockout (CRBNKO) H1299, and CRBNKO MCF7 cancer cells in response to TLR4 stimulation. Notably, we found that the ability of cancer migration and invasion was significantly enhanced in CRBNKO H1299 and CRBNKO MCF7 cancer cells, as compared with those of control cancer cells. Collectively, these results suggest that CRBN is a negative regulator of bactericidal activity and autophagy activation through inhibiting the TRAF6-induced ubiquitination of ECSIT and BECN1, respectively.

SUBMITTER: Kim MJ

PROVIDER: S-EPMC6759600 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1.

Kim Mi-Jeong MJ Min Yoon Y Shim Jae-Hyuck JH Chun Eunyoung E Lee Ki-Young KY

Frontiers in immunology 20190918

Cereblon (CRBN) as a multifunctional protein has been extensively studied. Here, we show that CRBN is a negative regulator of bactericidal activity and autophagy activation. Mitochondrial localization of CRBN was significantly increased in response to Toll-like receptor 4 (TLR4) stimulation. CRBN interrupted the association of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT)-TNF-receptor associated factor 6 (TRAF6) complex, thereby inhibiting the ubiquitination of ECSIT, ...[more]

PMID: 31620128

Similar Datasets

Project description:A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267-284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy.Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A1; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks' balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Project description:PurposeCisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines.Materials and methodsHuman BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation.ResultsCisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis.ConclusionCollectively, the study results indicated that cisplatin-induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin resistance related to autophagy induction.

Dataset Information

CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1.

Publications

CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets