ABSTRACT
Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.
Subject(s)
Apoptosis/drug effects , Caspase 10/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Interferon-gamma/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , CRISPR-Cas Systems/genetics , Caspase 10/chemistry , Caspase 10/genetics , Caspase 8/chemistry , Caspase 8/genetics , Caspase 8/metabolism , Caspase Inhibitors/pharmacology , Cell Line , Cytokine TWEAK/pharmacology , Drug Synergism , HT29 Cells , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Interferon-gamma/genetics , Interferon-gamma/metabolism , Mice , Mice, Knockout , Pentanoic Acids/pharmacology , Protein Kinases/deficiency , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/deficiency , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacologyABSTRACT
During immune responses, T cells are subject to clonal competition, which leads to the predominant expansion of high-affinity clones; however, there is little understanding of how this process is controlled. We found here that the transcription factor IRF4 was induced in a manner dependent on affinity for the T cell antigen receptor (TCR) and acted as a dose-dependent regulator of the metabolic function of activated T cells. IRF4 regulated the expression of key molecules required for the aerobic glycolysis of effector T cells and was essential for the clonal expansion and maintenance of effector function of antigen-specific CD8(+) T cells. Thus, IRF4 is an indispensable molecular 'rheostat' that 'translates' TCR affinity into the appropriate transcriptional programs that link metabolic function with the clonal selection and effector differentiation of T cells.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Interferon Regulatory Factors/metabolism , Orthomyxoviridae Infections/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/metabolism , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cell Differentiation , Cell Proliferation , Clone Cells , Gene Expression Regulation , Humans , Influenza A Virus, H3N2 Subtype/immunology , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Mice , Mice, Transgenic , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/virology , Transcription, GeneticABSTRACT
Regulatory T cells (T(reg) cells) are required for peripheral tolerance. Evidence indicates that T(reg) cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of T(reg) cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector T(reg) cells. Thus, our study defines a differentiation pathway that leads to the acquisition of T(reg) cell effector functions and requires both IRF4 and Blimp-1.
Subject(s)
Cell Differentiation/genetics , Interferon Regulatory Factors/genetics , T-Lymphocytes, Regulatory/metabolism , Transcription Factors/genetics , Animals , Base Sequence , Binding Sites/genetics , Cells, Cultured , Flow Cytometry , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Interferon Regulatory Factors/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Positive Regulatory Domain I-Binding Factor 1 , Protein Binding , Regulatory Sequences, Nucleic Acid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , T-Lymphocytes, Regulatory/cytology , Transcription Factors/metabolismSubject(s)
GTPase-Activating Proteins/metabolism , NF-kappa B/metabolism , Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Apoptosis Regulatory Proteins , Cell Cycle Proteins , GTPase-Activating Proteins/genetics , Mice , Mice, KnockoutABSTRACT
The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.
Subject(s)
Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/metabolism , NF-kappa B/genetics , Neoplasms/genetics , Ubiquitin/metabolism , Apoptosis/genetics , Carcinoma/genetics , Carcinoma/pathology , Cell Line , Cell Survival/genetics , Genes, Reporter , Glutathione Transferase/metabolism , Humans , Inhibitor of Apoptosis Proteins/genetics , Kidney/cytology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Luciferases/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Neoplasms/pathology , Plasmids , Protein Binding , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Transfection , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Ubiquitin/geneticsABSTRACT
Ubiquitylation is an essential cellular process, and yet many cancer cells appear to be more reliant upon it than normal cells as they are surprisingly sensitive to proteasome inhibitors (PI) and proteasome inhibitor drugs are well tolerated in vivo. Several reviews have suggested that specific protein targets account for PI induced cell death, but fail to adequately explain why cancer cells are more sensitive than normal cells to PIs. We review the evidence for these models, focusing primarily on inducers of cell death including p53 and the pro-apoptotic Bcl-2 Homology proteins (BH3 proteins) and propose an additional hypothesis; that a tumour cell's abnormal physiology makes it particularly reliant upon the proteasome. This hypothesis is well supported in the case of Multiple Myelomas, that may produce large amounts of antibodies and are therefore under considerable ER strain and in turn particularly reliant upon the proteasome to clear the large numbers of misfolded proteins. We propose that other cell types, tumor or non tumour, that are already under ER stress, or its equivalent, maybe particularly susceptible to proteasome inhibitors.