RESUMEN
Dynamic control of ubiquitination by deubiquitinating enzymes is essential for almost all biological processes. Ubiquitin-specific peptidase 22 (USP22) is part of the SAGA complex and catalyzes the removal of mono-ubiquitination from histones H2A and H2B, thereby regulating gene transcription. However, novel roles for USP22 have emerged recently, such as tumor development and cell death. Apart from apoptosis, the relevance of USP22 in other programmed cell death pathways still remains unclear. Here, we describe a novel role for USP22 in controlling necroptotic cell death in human tumor cell lines. Loss of USP22 expression significantly delays TNFα/Smac mimetic/zVAD.fmk (TBZ)-induced necroptosis, without affecting TNFα-mediated NF-κB activation or extrinsic apoptosis. Ubiquitin remnant profiling identified receptor-interacting protein kinase 3 (RIPK3) lysines 42, 351, and 518 as novel, USP22-regulated ubiquitination sites during necroptosis. Importantly, mutation of RIPK3 K518 reduced necroptosis-associated RIPK3 ubiquitination and amplified necrosome formation and necroptotic cell death. In conclusion, we identify a novel role of USP22 in necroptosis and further elucidate the relevance of RIPK3 ubiquitination as crucial regulator of necroptotic cell death.
Asunto(s)
Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Ubiquitina Tiolesterasa , Apoptosis/genética , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer's disease.
Asunto(s)
Proteómica , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Transducción de SeñalRESUMEN
Ubiquitin-specific peptidase 22 (USP22) is a deubiquitinating enzyme (DUB) that underlies tumorigenicity, proliferation, cell death and differentiation through deubiquitination of histone and non-histone targets. Ubiquitination determines stability, localization and functions of cell fate proteins and controls cell-protective signaling pathways to surveil cell cycle progression. In a variety of carcinomas, lymphomas and leukemias, ubiquitination regulates the tumor-suppressive functions of the promyelocytic leukemia protein (PML), but PML-specific DUBs, DUB-controlled PML ubiquitin sites and the functional consequences of PML (de)ubiquitination remain unclear. Here, we identify USP22 as regulator of PML and the oncogenic acute promyelocytic leukemia (APL) fusion PML-RARα protein stability and identify a destabilizing role of PML residue K394. Additionally, loss of USP22 upregulates interferon (IFN) and IFN-stimulated gene (ISG) expression in APL and induces PML-RARα stabilization and a potentiation of the cell-autonomous sensitivity towards all-trans retinoic acid (ATRA)-mediated differentiation. Our findings imply USP22-dependent surveillance of PML-RARα stability and IFN signaling as important regulator of APL pathogenesis, with implications for viral mimicry, differentiation and cell fate regulation in other leukemia subtypes.
RESUMEN
Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endo- and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in cells of human origin. Loss of LUBAC activity inhibits tumor necrosis factor α (TNFα)-mediated necroptosis, not by affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Finally, we confirm LUBAC-dependent activation of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as novel regulator of necroptosis which promotes MLKL membrane accumulation in human cells and pioneer primary human organoids to model necroptosis in near-physiological settings.
Asunto(s)
Necroptosis , Proteínas Quinasas , Humanos , Necrosis/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Fosforilación , Muerte Celular , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Apoptosis/fisiologíaRESUMEN
Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFNs (IFN-λ) exhibit cell-type specific and long-lasting functions in auto-inflammation, tumorigenesis, and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2'3'-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication, and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.
Asunto(s)
COVID-19 , Interferón Tipo I , Proteínas de la Membrana/metabolismo , Ubiquitina Tiolesterasa , Antivirales/farmacología , Humanos , Interferón Tipo I/genética , Interferones/metabolismo , Pandemias , SARS-CoV-2 , Ubiquitina Tiolesterasa/metabolismo , Interferón lambdaRESUMEN
Burkitt's lymphoma (BL) is a highly aggressive form of B-cell non-Hodgkin's lymphoma. The clinical outcome in children with BL has improved over the last years but the prognosis for adults is still poor, highlighting the need for novel treatment strategies. Here, we report that the combinational treatment with the Smac mimetic BV6 and TRAIL triggers necroptosis in BL when caspases are blocked by zVAD.fmk (TBZ treatment). The sensitivity of BL cells to TBZ correlates with MLKL expression. We demonstrate that necroptotic signaling critically depends on MLKL, since siRNA-induced knockdown and CRISPR/Cas9-mediated knockout of MLKL profoundly protect BL cells from TBZ-induced necroptosis. Conversely, MLKL overexpression in cell lines expressing low levels of MLKL leads to necroptosis induction, which can be rescued by pharmacological inhibitors, highlighting the important role of MLKL for necroptosis execution. Importantly, the methylation status analysis of the MLKL promoter reveals a correlation between methylation and MLKL expression. Thus, MLKL is epigenetically regulated in BL and might serve as a prognostic marker for treatment success of necroptosis-based therapies. These findings have crucial implications for the development of new treatment options for BL.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Proteínas Mitocondriales/metabolismo , Necroptosis/genética , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Mimetismo Biológico , Linfoma de Burkitt/patología , Muerte Celular , Línea Celular Tumoral , Susceptibilidad a Enfermedades , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Proteínas Mitocondriales/química , Necroptosis/efectos de los fármacos , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Interferencia de ARNRESUMEN
Adhesion G protein-coupled receptors (ADGRs) have recently become a target of intense research. Their unique protein structure, which consists of a G protein-coupled receptor combined with long adhesive extracellular domains, suggests a dual role in cell signaling and adhesion. Despite considerable progress in the understanding of ADGR signaling over the past years, the knowledge about ADGR protein networks is still limited. For most receptors, only a few interaction partners are known thus far. We aimed to identify novel ADGR-interacting partners to shed light on cellular protein networks that rely on ADGR function. For this, we applied affinity proteomics, utilizing tandem affinity purifications combined with mass spectrometry. Analysis of the acquired proteomics data provides evidence that ADGRs not only have functional roles at synapses but also at intracellular membranes, namely at the endoplasmic reticulum, the Golgi apparatus, mitochondria, and mitochondria-associated membranes (MAMs). Specifically, we found an association of ADGRs with several scaffold proteins of the membrane-associated guanylate kinases family, elementary units of the γ-secretase complex, the outer/inner mitochondrial membrane, MAMs, and regulators of the Wnt signaling pathways. Furthermore, the nuclear localization of ADGR domains together with their physical interaction with nuclear proteins and several transcription factors suggests a role of ADGRs in gene regulation.