RESUMEN
Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1ß-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.
Asunto(s)
Retroelementos , Animales , Humanos , Ratones , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Retrovirus Endógenos/genética , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Histonas/genética , Elementos de Nucleótido Esparcido Largo/genética , Unión Proteica , Retroelementos/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Complejos Multiproteicos/metabolismoRESUMEN
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
The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the Ë29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
Asunto(s)
Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas Cullin/metabolismo , Humanos , Modelos Moleculares , Mutación , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.
Asunto(s)
Linaje de la Célula/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Homólogo de la Proteína Chromobox 5 , Eucromatina/genética , Eucromatina/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Nucleosomas/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Transcripción GenéticaRESUMEN
Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called "microgliopathies". However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin-specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon-induced genes, thereby terminating IFN signaling. The Usp18-mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non-diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy.
Asunto(s)
Encéfalo/metabolismo , Endopeptidasas/deficiencia , Interferones/metabolismo , Microglía/metabolismo , Modelos Neurológicos , Transducción de Señal/fisiología , Animales , Western Blotting , Clonación Molecular , Cartilla de ADN/genética , Endopeptidasas/genética , Endopeptidasas/metabolismo , Técnicas Histológicas , Ratones , Ratones Noqueados , Análisis por Micromatrices , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Estadísticas no Paramétricas , Ubiquitina TiolesterasaRESUMEN
Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18(C61A/C61A)) expressing enzymatically inactive USP18. USP18(C61A/C61A) mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18(-/-) mice, USP18(C61A/C61A) animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18(C61A/C61A) mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18(C61A/C61A) mice was completely reversed in USP18(C61A/C61A) mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects.
Asunto(s)
Citocinas/metabolismo , Farmacorresistencia Viral , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Animales , Antivirales/farmacología , Células Cultivadas , Virus de la Influenza B/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ubiquitinas/metabolismoRESUMEN
Posttranslational protein modification by ubiquitin and ubiquitin-like modifiers (UBLs) is mediated by a hierarchical cascade of conjugating enzymes and affects multiple biological processes within the cell. Interferon-stimulated gene 15 (ISG15) is an UBL, which is strongly induced by type I Interferon and ISG15 modification was shown to play an essential role in antiviral defense. While hHERC5 is the major E3 ligase for ISG15 modification in humans, ISGylation in the murine systems at the level of E3 ligases was weakly characterized as rodent genomes lack a direct homologue of hHERC5. Here, we show that mHERC6 is strongly induced by different pathogen-associated molecular patterns (PAMPs) in a type I Interferon receptor (IFNAR1) dependent manner. We demonstrate that mHERC6 is essential for endogenous murine ISGylation and thus represents the dominant ISG15 E3 ligase in mice. In contrast to its human homologue, mHERC6 is also capable to mediate conjugation of human ISG15.
Asunto(s)
Citocinas/metabolismo , Interacciones Huésped-Patógeno , Procesamiento Proteico-Postraduccional , Receptor de Interferón alfa y beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Ubiquitina-Proteína Ligasas/genética , Ubiquitinas/metabolismoRESUMEN
BACKGROUND: Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens. RESULTS: High-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone. CONCLUSION: The results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.
Asunto(s)
Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Escherichia coli/genética , Expresión Génica , Animales , Proteínas de Escherichia coli/metabolismo , Fibroblastos/metabolismo , Ratones , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Ubiquitina TiolesterasaRESUMEN
The ubiquitin-specific protease 18 (USP18) has two major functions: (a) it is a highly specific protease that cleaves the ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) from proteins, and (b) independent from its enzymatic activity USP18 interacts with the type I interferon receptor and shuts off downstream signaling. The structures of USP18 and a USP18-ISG15 complex revealed the molecular basis of the unique specificity of the protease and might shed some light into its interaction with the interferon receptor.
Asunto(s)
Citocinas/metabolismo , Endopeptidasas/metabolismo , Transducción de Señal , Ubiquitinas/metabolismo , Citocinas/química , Endopeptidasas/química , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Proteolisis , Receptor de Interferón alfa y beta/metabolismo , Especificidad por Sustrato , Ubiquitina Tiolesterasa , Ubiquitinas/químicaRESUMEN
Ubiquitin-specific proteases (USPs) represent the largest family of deubiquitinating enzymes (DUB). These proteases cleave the isopeptide bond between ubiquitin and a lysine residue of a ubiquitin-modified protein. USP18 is a special member of the USP family as it only deconjugates the ubiquitin-like protein ISG15 (interferon-stimulated gene (ISG) 15) from target proteins but is not active towards ubiquitin. Independent of its protease activity, USP18 functions as a major negative regulator of the type I interferon response showing that USP18 is - at least - a bifunctional protein. In this review, we summarise our current knowledge of protease-dependent and -independent functions of USP18 and discuss the structural basis of its dual activity.
Asunto(s)
Endopeptidasas/química , Interferón Tipo I/química , Péptido Hidrolasas/química , Ubiquitina/genética , Citocinas/química , Citocinas/genética , Endopeptidasas/genética , Humanos , Interferón Tipo I/genética , Péptido Hidrolasas/genética , Conformación Proteica , Proteolisis , Transducción de Señal/genética , Ubiquitina/química , Ubiquitina Tiolesterasa , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.
Asunto(s)
Citocinas/metabolismo , Endopeptidasas/química , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Proteínas de Pez Cebra/química , Animales , Cristalización , Cristalografía por Rayos X , Citocinas/química , Endopeptidasas/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Relación Estructura-Actividad , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitinas/química , Ubiquitinas/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 ± 0.2 µm. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 ± 0.2 µm and a kcat of 0.23 ± 0.004 s(-1) , respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. STRUCTURED DIGITAL ABSTRACT: âISG15 and USP18 bind by microscale thermophoresis (View interaction) âUSP18 cleaves ISG15 by enzymatic study (View interaction).