RESUMEN
The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3ß. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Endopeptidasas/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Timocitos/inmunología , Ubiquitina Tiolesterasa/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Colitis/genética , Colitis/inmunología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Humanos , Células Jurkat , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-7/inmunología , Receptores de Interleucina-7/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timocitos/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) also called ubiquitin D (UBD) is a member of the ubiquitin-like modifier (ULM) family. The FAT10 gene is localized in the MHC class I locus and FAT10 protein expression is mainly restricted to cells and organs of the immune system. In all other cell types and tissues, FAT10 expression is highly inducible by the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF). Besides ubiquitin, FAT10 is the only ULM which directly targets its substrates for degradation by the 26S proteasome. This poses the question as to why two ULMs sharing the proteasome-targeting function have evolved and how they differ from each other. This Review summarizes the current knowledge of the special structure of FAT10 and highlights its differences from ubiquitin. We discuss how these differences might result in differential outcomes concerning proteasomal degradation mechanisms and non-covalent target interactions. Moreover, recent insights about the structural and functional impact of FAT10 interacting with specific non-covalent interaction partners are reviewed.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitina , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Factor de Necrosis Tumoral alfa , Ubiquitinas/genéticaRESUMEN
The retina-specific chaperone aryl hydrocarbon interacting protein-like 1 (AIPL1) is essential for the correct assembly of phosphodiesterase 6 (PDE6), which is a pivotal effector enzyme for phototransduction and vision because it hydrolyzes cGMP. AIPL1 interacts with the cytokine-inducible ubiquitin-like modifier FAT10, which gets covalently conjugated to hundreds of proteins and targets its conjugation substrates for proteasomal degradation, but whether FAT10 affects PDE6 function or turnover is unknown. Here, we show that FAT10 mRNA is expressed in human retina and identify rod PDE6 as a retina-specific substrate of FAT10 conjugation. We found that AIPL1 stabilizes the FAT10 monomer and the PDE6-FAT10 conjugate. Additionally, we elucidated the functional consequences of PDE6 FAT10ylation. On the one hand, we demonstrate that FAT10 targets PDE6 for proteasomal degradation by formation of a covalent isopeptide linkage. On the other hand, FAT10 inhibits PDE6 cGMP hydrolyzing activity by noncovalently interacting with the PDE6 GAFa and catalytic domains. Therefore, FAT10 may contribute to loss of PDE6 and, as a consequence, degeneration of retinal cells in eye diseases linked to inflammation and inherited blindness-causing mutations in AIPL1.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Retina/metabolismo , Ubiquitinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Dominio Catalítico , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/química , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Unión Proteica , Proteolisis/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ubiquitina/metabolismo , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
The deubiquitylation of target proteins is mediated by deubiquitylating enzymes (DUB) such as OTUB1, which plays an important role in immune response, cell cycle progression, and DNA repair. Within these processes, OTUB1 reduces the ubiquitylation of target proteins in two distinct ways, either by using its catalytic DUB activity or in a noncatalytic manner by inhibiting the E2-conjugating enzyme. Here, we show that the ubiquitin-like modifier FAT10 regulates OTUB1 stability and functionality in different ways. Covalent FAT10ylation of OTUB1 resulted in its proteasomal degradation, whereas a noncovalent interaction stabilized OTUB1. We provide evidence that OTUB1 interacts directly with FAT10 and the E2-conjugating enzyme USE1. This interaction strongly stimulated OTUB1 DUB activity toward Lys-48-linked diubiquitin. Furthermore, the noncovalent interaction between FAT10 and OTUB1 not only enhanced its isopeptidase activity toward Lys-48-linked ubiquitin moieties but also strengthened its noncatalytic activity in reducing Lys-63 polyubiquitylation of its target protein TRAF3 (TNF receptor-associated factor 3). Additionally, the cellular clearance of overall polyubiquitylation by OTUB1 was strongly stimulated through the presence of FAT10. The addition of FAT10 also led to an increased interaction between OTUB1 and its cognate E2 UbcH5B, implying a function of FAT10 in the inhibition of polyubiquitylation. Overall, these data indicate that FAT10 not only plays a role in covalent modification, leading its substrates to proteasomal degradation, but also regulates the stability and functionality of target proteins by interacting in a noncovalent manner. FAT10 is thereby able to exert a major influence on ubiquitylation processes.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Enzimas Desubicuitinizantes , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Proteínas SNARE , Ubiquitinación , Proteínas de Transporte VesicularRESUMEN
Biallelic mutations in the photoreceptor-expressed aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) are associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy in early childhood. AIPL1 functions as a photoreceptor-specific co-chaperone that interacts with the molecular chaperone HSP90 to facilitate the stable assembly of the retinal cyclic GMP (cGMP) phosphodiesterase (PDE6) holoenzyme. In this study, we characterized the functional deficits of AIPL1 variations, some of which induce aberrant pre-mRNA AIPL1 splicing leading to the production of alternative AIPL1 isoforms. We investigated the ability of the AIPL1 variants to mediate an interaction with HSP90 and modulate the rod cGMP PDE6 stability and activity. Our data revealed that both the FK506 binding protein (FKBP)-like domain and the tetratricopeptide repeat (TPR) domain of AIPL1 are required for interaction with HSP90. We further demonstrate that AIPL1 significantly modulates the catalytic activity of heterologously expressed rod PDE6. Although the N-terminal FKBP-like domain of AIPL1 binds the farnesylated PDE6α subunit through direct interaction with the farnesyl moiety, mutations compromising the integrity of the C-terminal TPR domain of AIPL1 also failed to modulate PDE6 activity efficiently. These AIPL1 variants moreover failed to promote the HSP90-dependent stabilization of the PDE6α subunit in the cytosol. In summary, we have successfully validated the disease-causing status of the AIPL1 variations in vitro. Our findings provide insight into the mechanism underlying the co-chaperone role of AIPL1 and will be critical for ensuring an early and effective diagnosis of AIPL1 LCA patients.
Asunto(s)
Proteínas Portadoras/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Proteínas del Ojo/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células CHO , Proteínas Portadoras/química , Cricetulus , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/genética , Células HEK293 , Proteínas HSP90 de Choque Térmico/química , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Mutación , Unión Proteica , Dominios Proteicos , Precursores del ARN/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiología , Relación Estructura-ActividadRESUMEN
The TNF and IFN-γ-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) is most prominently expressed in immunological tissues but information regarding basal expression and inducibility of FAT10 in the different types of immune cells is still lacking. Hence, we investigated FAT10 mRNA expression in the major human and murine immune cell subsets, and FAT10 protein expression in human leukocytes. We isolated the different human leukocytes from peripheral blood and the murine immune cell subsets from spleen. The purified leukocytes were left untreated or stimulated with TNF and INF-γ or LPS to induce FAT10 followed by quantitative real-time PCR or western blot analysis. Basal expression of FAT10 mRNA and protein was generally low but strongly up-regulated by IFN-γ and TNF in all immune cell subsets. LPS treatment induced FAT10 expression marginally in human CD8+ T cells and murine granulocytes, but it increased Fat10 expression significantly in murine regulatory T cells. Yet, in human CD8+ T cells, natural killer cells, natural killer T cells, and dendritic cells, the FAT10 mRNA was expressed without induction. Similarly, murine macrophages, monocytes, and regulatory T cells expressed Fat10 in the absence of stimulation. In summary, our findings suggest particular functions of FAT10 in these cell types. Furthermore, we observed not only a cell type-specific but also a species-specific basal FAT10 expression profile. Our data will serve as a guideline for future investigations to further elucidate FAT10's role in the immune system.
Asunto(s)
Leucocitos/metabolismo , Ubiquitinas/genética , Ubiquitinas/fisiología , Animales , Células HEK293 , Humanos , Interferón gamma/metabolismo , Leucocitos/inmunología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/genética , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinas/metabolismo , Regulación hacia ArribaRESUMEN
Bacterial invasion of eukaryotic cells is counteracted by cell-autonomous innate immune mechanisms including xenophagy. The decoration of cytosolic bacteria by ubiquitylation and binding of galectin-8 leads to recruitment of autophagy adaptors like p62 (also known as SQSTM1), NDP52 (also known as CALCOCO2) and optineurin, which initiate the destruction of bacteria by xenophagy. Here, we show that the functionally barely characterized IFNγ- and TNFα-inducible ubiquitin-like modifier FAT10 (also known as ubiquitin D, UBD), which binds to the autophagy adaptor p62, but has not been shown to associate with pathogens before, is recruited to cytosolic Salmonella Typhimurium in human cells. FAT10-decorated S. Typhimurium were simultaneously decorated with ubiquitin, p62, NDP52 and the autophagy marker LC3B (MAP1LC3B). FAT10 colocalized with p62-positive microdomains on S. Typhimurium, whereas colocalization with NDP52 was only partial. A kinetic analysis revealed an early, but only transient, decoration of bacteria by FAT10, which resembled that of p62. Although bacterial replication was not detectably altered in FAT10-depleted or overexpressing cells in vitro, survival experiments revealed that NRAMP1-transgenic mice that were FAT10-deficient had a higher susceptibility to orally inoculated S. Typhimurium bacteria than NRAMP1-transgenic mice that were wild-type for FAT10. Taken together, our data suggest a role for FAT10 in the intracellular defense against bacteria.
Asunto(s)
Salmonella typhimurium/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animales , Autofagia , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
The Nobel prize has been awarded for the discovery of ubiquitin as a transferable signal for the degradation of proteins by the 26S proteasome. While isopeptide linkage of a protein with a single ubiquitin does not serve as a degradation signal for the proteasome, poly-ubiquitylation via several different lysine residues within ubiquitin leads to efficient proteasomal degradation. Ubiquitin-like modifiers have not been shown to directly mediate proteasomal degradation except for the cytokine inducible modifier HLA-F adjacent transcript 10 (FAT10), which consists of two ubiquitin-like domains. FAT10 ends with a free diglycine motif at its C-terminus which is required for isopeptide linkage to hundreds of different substrates. In contrast to ubiquitin, a single FAT10 suffices to bind to the 26S proteasome and to efficiently mediate proteasomal degradation in a ubiquitin-independent manner. Here we review the data on ubiquitin-independent degradation by FAT10, on how FAT10 is conjugated to its substrates, how FAT10 binds to the 26S proteasome, and how the ubiquitin-like (UBL)-ubiquitin-associated (UBA) protein NUB1L accelerates FAT10 mediated proteolysis. Finally, with a glimpse on recently identified substrates, we will discuss the currently emerging knowledge about the biological functions of FAT10. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitinación/fisiología , Ubiquitinas/metabolismo , Animales , Humanos , Señales de Clasificación de Proteína/fisiologíaRESUMEN
FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-κB activation. Upon induction by pro-inflammatory cytokines, hundreds of endogenous substrates become covalently conjugated to FAT10 leading to their proteasomal degradation. Nevertheless, only three substrates have been identified so far to which FAT10 becomes covalently attached through a non-reducible isopeptide bond, and these are the FAT10-conjugating enzyme USE1 which auto-FAT10ylates itself in cis, the tumor suppressor p53 and the ubiquitin-activating enzyme UBE1 (UBA1). To identify additional FAT10 substrates and interaction partners, we used a new monoclonal FAT10-specific antibody to immunopurify endogenous FAT10 conjugates from interferon (IFN)γ-and tumor necrosis factor (TNF)α-stimulated cells for identification by mass spectrometry. In addition to two already known FAT10-interacting proteins, histone deacetylase 6 and UBA6, we identified 569 novel FAT10-interacting proteins involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer. Thirty-one percent of all identified proteins were categorized as putative covalently linked substrates. One of the identified proteins, the autophagosomal receptor p62/SQSTM1, was further investigated. p62 becomes covalently mono-FAT10ylated at several lysines, and FAT10 colocalizes with p62 in p62 bodies. Strikingly, FAT10ylation of p62 leads to its proteasomal degradation, and prolonged induction of endogenous FAT10 expression by pro-inflammatory cytokines leads to a decrease of endogenous p62. The elucidation of the FAT10 degradome should enable a better understanding of why FAT10 has evolved as an additional transferable tag for proteasomal degradation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteómica , Ubiquitinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Autofagia/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Interferón gamma/farmacología , Lisina/metabolismo , Espectrometría de Masas , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteína Sequestosoma-1 , Especificidad por Sustrato/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The ubiquitin-like modifier FAT10 is upregulated under pro-inflammatory conditions, targets its substrates for proteasomal degradation and functions as a negative regulator of the type-I IFN response. Influenza A virus infection upregulates the production of type-I IFN and the expression of the E3 ligase TRIM21, which regulates type-I IFN production in a positive feedback manner. In this study, we show that FAT10 becomes covalently conjugated to TRIM21 and that this targets TRIM21 for proteasomal degradation. We further show that the coiled-coil and PRYSPRY domains of TRIM21 and the C-terminal diglycine motif of FAT10 are important for the TRIM21-FAT10 interaction. Moreover, upon influenza A virus infection and in the presence of FAT10 the total ubiquitination of TRIM21 is reduced and our data reveal that the FAT10-mediated degradation of TRIM21 diminishes IFNß production. Overall, this study provides strong evidence that FAT10 down-regulates the antiviral type-I IFN production by modulating additional molecules of the RIG-I signaling pathway besides the already published OTUB1. In addition, we elucidate a novel mechanism of FAT10-mediated proteasomal degradation of TRIM21 that regulates its stability.
Asunto(s)
Interferón Tipo I , Complejo de la Endopetidasa Proteasomal , Ribonucleoproteínas , Ubiquitinación , Ubiquitinas , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Interferón Tipo I/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación hacia Abajo , Células HEK293 , Transducción de Señal , Virus de la Influenza A/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , AnimalesRESUMEN
IFN-I secretion provides a rapid host defense against infection with RNA viruses. Within the host cell, viral RNA triggers the activation of the RIG-I signaling pathway, leading to the production of IFN-I. Because an exaggerated IFN-I response causes severe tissue damage, RIG-I signaling is tightly regulated. One of the factors that control the IFN-I response is the ubiquitin-like modifier FAT10, which is induced by TNF and IFNγ and targets covalently FAT10-linked proteins for proteasomal degradation. However, the mechanism of how FAT10 modulates IFN-I secretion remains to be fully elucidated. Here, we provide strong evidence that FAT10 is phosphorylated by IκB kinase ß (IKKß) upon TNF stimulation and during influenza A virus infection on several serine and threonine residues. FAT10 phosphorylation increases the binding of FAT10 to the TRAF3-deubiquitylase OTUB1 and its FAT10-mediated activation. Consequently, FAT10 phosphorylation results in a low ubiquitylation state of TRAF3, which is unable to maintain interferon regulatory factor 3 phosphorylation and downstream induction of IFN-I. Taken together, we reveal a mechanism of how phosphorylation of FAT10 limits the production of tissue-destructive IFN-I in inflammation.
Asunto(s)
Quinasa I-kappa B , Interferón Tipo I , Factor 3 Asociado a Receptor de TNF , Proteínas Serina-Treonina Quinasas , AntiviralesRESUMEN
BACKGROUND: Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE: Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS: CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS: A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS: Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.
Asunto(s)
Agammaglobulinemia/genética , Agammaglobulinemia/inmunología , Linfocitos B/metabolismo , Infecciones/inmunología , Receptores de Complemento 3d/metabolismo , Adulto , Agammaglobulinemia/complicaciones , Agammaglobulinemia/diagnóstico , Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Señalización del Calcio/genética , Complemento C3d/metabolismo , Análisis Mutacional de ADN , Células HEK293 , Humanos , Inmunidad Humoral/genética , Memoria Inmunológica/genética , Infecciones/diagnóstico , Infecciones/etiología , Infecciones/genética , Masculino , Unión Proteica/genética , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/inmunología , Eliminación de Secuencia/genética , Transgenes/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
The ubiquitin-like modifier FAT10 is up-regulated in many different cell types by IFNγ and TNFα (TNF) and directly targets proteins for proteasomal degradation. FAT10 gets covalently conjugated to its conjugation substrates by the E1 activating enzyme UBA6, the E2 conjugating enzyme USE1, and E3 ligases including Parkin. To date, USE1 was supposed to be the only E2 enzyme for FAT10ylation, and we show here that a knockout of USE1 strongly diminished FAT10 conjugation. Remarkably, under inflammatory conditions in the presence of TNF, FAT10 conjugation appears to be independent of USE1. We report on the identification of additional E2 conjugating enzymes, which were previously not associated with FAT10. We confirm their capacity to be charged with FAT10 onto their active site cysteine, and to rescue FAT10 conjugation in the absence of USE1. This finding strongly widens the field of FAT10 research by pointing to multiple, so far unknown pathways for the conjugation of FAT10, disclosing novel possibilities for pharmacological interventions to regulate FAT10 conjugation under inflammatory conditions and/or viral infections.
Asunto(s)
Inflamación , Factor de Necrosis Tumoral alfa , Ubiquitina , Cisteína , Ubiquitina/metabolismo , Humanos , Inflamación/metabolismoRESUMEN
The ubiquitin-like modifier FAT10 is highly upregulated under inflammatory conditions and targets its conjugation substrates to the degradation by the 26S proteasome. This process termed FAT10ylation is mediated by an enzymatic cascade and includes the E1 activating enzyme ubiquitin-like modifier activating enzyme 6 (UBA6), the E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1) and E3 ligases, such as Parkin. In this study, the function of the HECT-type ubiquitin E3 ligase HUWE1 was investigated as a putative E3 ligase and/or conjugation substrate of FAT10. Our data provide strong evidence that HUWE1 is FAT10ylated in a UBA6 and FAT10 diglycine-dependent manner in vitro and in cellulo and that the HUWE1-FAT10 conjugate is targeted to proteasomal degradation. Since the mutation of all relevant cysteine residues within the HUWE1 HECT domain did not abolish FAT10 conjugation, a role of HUWE1 as E3 ligase for FAT10ylation is rather unlikely. Moreover, we have identified the autophagy-related protein AMBRA1 as a new FAT10 interaction partner. We show that the HUWE1-FAT10 conjugate formation is diminished in presence of AMBRA1, while the interaction between AMBRA1 and HUWE1 is strengthened in presence of FAT10. This implies a putative interplay of all three proteins in cellular processes such as mitophagy.
Asunto(s)
Ubiquitina , Ubiquitinas , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The interaction of the 19S regulatory particle of the 26S proteasome with ubiquitylated proteins leads to gate opening of the 20S core particle and increases its proteolytic activity by binding of the ubiquitin chain to the inhibitory deubiquitylation enzyme USP14 on the 19S regulatory subunit RPN1. Covalent modification of proteins with the cytokine inducible ubiquitin-like modifier FAT10 is an alternative signal for proteasomal degradation. Here, we report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. We also show that FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the subunit RPN1. In conclusion, the herein described cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome.
Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitinas , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , AnimalesRESUMEN
Parkin is an E3 ubiquitin ligase belonging to the RING-between-RING family. Mutations in the Parkin-encoding gene PARK2 are associated with familial Parkinson's disease. Here, we investigate the interplay between Parkin and the inflammatory cytokine-induced ubiquitin-like modifier FAT10. FAT10 targets hundreds of proteins for degradation by the 26S proteasome. We show that FAT10 gets conjugated to Parkin and mediates its degradation in a proteasome-dependent manner. Parkin binds to the E2 enzyme of FAT10 (USE1), auto-FAT10ylates itself, and facilitates FAT10ylation of the Parkin substrate Mitofusin2 in vitro and in cells, thus identifying Parkin as a FAT10 E3 ligase. On mitochondrial depolarization, FAT10ylation of Parkin inhibits its activation and ubiquitin-ligase activity causing impairment of mitophagy progression and aggravation of rotenone-mediated death of dopaminergic neuronal cells. In conclusion, FAT10ylation inhibits Parkin and mitophagy rendering FAT10 a likely inflammation-induced exacerbating factor and potential drug target for Parkinson's disease.
Asunto(s)
Mitofagia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Muerte Celular , Citosol/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Neuronas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Especificidad por Sustrato , UbiquitinaciónRESUMEN
Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21-shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation.
Asunto(s)
Citoplasma/metabolismo , Receptores de Complemento 3d/química , Receptores de Complemento 3d/metabolismo , Secuencia de Aminoácidos , Animales , Antioxidantes/farmacología , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Citoplasma/efectos de los fármacos , Fluorescencia , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Eliminación de Secuencia , Compuestos de Sulfhidrilo/farmacología , Factores de Tiempo , Vanadatos/farmacologíaRESUMEN
The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.
Asunto(s)
Ubiquitinas/metabolismo , Western Blotting , Línea Celular , Expresión Génica , Humanos , Inmunoprecipitación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección/métodos , Ubiquitinas/genética , Ubiquitinas/aislamiento & purificación , Regulación hacia ArribaRESUMEN
The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation.