ABSTRACT
The inflammasome is a major component of the innate immune system, and its main function is to activate caspase-1, a cysteine protease that promotes inflammation by inducing interleukin-1ß (IL-1ß) maturation and release into the extracellular milieu. To prevent uncontrolled inflammation, this complex is highly regulated. When it is assembled, the inflammasome is insoluble, which has long precluded the analysis of its interactions with other proteins. Here we used the proximity-dependent biotinylation assay (BioID) to identify proteins associated with caspase-1 during inflammasome activation. Using the BioID in a cell-free system in which the inflammasome had been activated, we found that a caspase-1-biotin ligase fusion protein selectively labeled 111 candidates, including the p62/sequestosome-1 protein (p62). Using co-immunoprecipitation experiments, we demonstrated that p62 interacts with caspase-1. This interaction promoted caspase-1-mediated cleavage of p62 at Asp-329. Mechanistic and functional analyses revealed that caspase-1-mediated cleavage of p62 leads to loss of its interaction with the autophagosomal protein microtubule-associated protein 1 light chain 3 ß (LC3B). Strikingly, overexpression of a p62 N-terminal fragment generated upon caspase-1 cleavage decreased IL-1ß release, whereas overexpression of p62's C-terminal portion enhanced IL-1ß release, by regulating pro-IL1ß levels. Overall, the overexpression of both fragments together decreased IL-1ß release. Taken together, our results indicate that caspase-1-mediated p62 cleavage plays a complex role in balancing caspase-1-induced inflammation.
Subject(s)
Apoptosis , Caspase 1/metabolism , Inflammasomes , Interleukin-1beta/metabolism , Sequestosome-1 Protein/metabolism , Staining and Labeling/methods , Animals , Biological Assay , Biotinylation , Caspase 1/genetics , HEK293 Cells , Humans , Mice , Sequestosome-1 Protein/geneticsABSTRACT
The endoplasmic reticulum kinase inositol-requiring enzyme 1 (IRE1) and its downstream target X-box-binding protein 1 (XBP1) drive B-cell differentiation toward plasma cells and have been shown to contribute to multiple myeloma development; yet, little is known of the role of this pathway in diffuse large B-cell lymphoma (DLBCL). Here, we show that in the germinal center B-cell-like (GCB) DLBCL subtype, IRE1 expression is reduced to a level that prevents XBP1 activation. Gene expression profiles indicated that, in GCB DLBCL cancer samples, expression of IRE1 messenger RNA was inversely correlated with the levels and activity of the epigenetic repressor, histone methyltransferase enhancer of zeste homolog 2 (EZH2). Correspondingly, in GCB-derived cell lines, the IRE1 promoter carried increased levels of the repressive epigenetic mark histone 3 lysine 27 trimethylation. Pharmacological inhibition of EZH2 erased those marks and restored IRE1 expression and function in vitro and in vivo. Moreover, reconstitution of the IRE1-signaling pathway, by expression of the XBP1-active form, compromised GCB DLBCL tumor growth in a mouse xenograft cancer model. These findings indicate that IRE1-XBP1 downregulation distinguishes GCB DLBCL from other DLBCL subtypes and contributes to tumor growth.
Subject(s)
B-Lymphocytes/immunology , Endoribonucleases/genetics , Gene Expression Regulation, Neoplastic , Germinal Center/immunology , Lymphoma, Large B-Cell, Diffuse/immunology , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , X-Box Binding Protein 1/genetics , Animals , B-Lymphocytes/pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Endoribonucleases/antagonists & inhibitors , Endoribonucleases/immunology , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/immunology , Epigenesis, Genetic , Germinal Center/pathology , Histones/genetics , Histones/immunology , Humans , Indazoles/pharmacology , Leupeptins/pharmacology , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoma, Large B-Cell, Diffuse/therapy , Mice , Mice, 129 Strain , Plasma Cells/immunology , Plasma Cells/pathology , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/immunology , Pyridones/pharmacology , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/immunology , Signal Transduction , X-Box Binding Protein 1/antagonists & inhibitors , X-Box Binding Protein 1/immunology , Xenograft Model Antitumor AssaysABSTRACT
Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.