RESUMEN
The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Ubiquitina/metabolismo , Animales , Sitios de Unión , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/genética , Cristalografía por Rayos X , Citocinas/genética , Evaluación Preclínica de Medicamentos/métodos , Reposicionamiento de Medicamentos , Polarización de Fluorescencia , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Inhibidores de Proteasas/farmacología , Conformación Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Ubiquitinas/genética , Células VeroRESUMEN
The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.
Asunto(s)
Activación Enzimática/fisiología , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/metabolismo , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , TransfecciónRESUMEN
Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Shigella flexneri/enzimología , Shigella flexneri/inmunología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Amidohidrolasas/genética , Secuencia de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Biocatálisis , Caspasas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Cisteína/metabolismo , Análisis Mutacional de ADN , Diglicéridos/antagonistas & inhibidores , Diglicéridos/metabolismo , Disentería Bacilar/microbiología , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Células HEK293 , Células HeLa , Histidina/metabolismo , Humanos , Inmunidad Innata , Inflamación/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética , Factores de Virulencia/metabolismoRESUMEN
We report a case of severe Aicardi-Goutières syndrome caused by a novel homozygous RNASEH2B intronic variant, NC_000013.10(NM_024570.4):c.65-13G > A p.Glu22Valfs*5. The patient was born with pseudo-TORCH symptoms, including intracranial calcification, cataracts, and hepatosplenomegaly. Furthermore, the patient exhibited profound intellectual impairment and died at 14 months due to aspiration pneumonia accompanied by interstitial lung abnormalities. The severity of the patient's symptoms underscores the critical role of the C-terminal region of RNase H2B.
RESUMEN
OTU deubiquitinase with linear linkage specificity (OTULIN) regulates inflammation and cell death by deubiquitinating linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC). Biallelic loss-of-function mutations causes OTULIN-related autoinflammatory syndrome (ORAS), while OTULIN haploinsuffiency has not been associated with spontaneous inflammation. However, herein, we identify two patients with the heterozygous mutation p.Cys129Ser in OTULIN. Consistent with ORAS, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF-induced death, and dysregulation of inflammatory signaling in patient cells. While the C129S mutation did not affect OTULIN protein stability or binding capacity to LUBAC and linear ubiquitin chains, it did ablate OTULIN deubiquitinase activity. Loss of activity facilitated the accumulation of autoubiquitin chains on LUBAC. Altered ubiquitination of LUBAC inhibits its recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death and disease pathology. By reporting the first dominant negative mutation driving ORAS, this study expands our clinical understanding of OTULIN-associated pathology.
Asunto(s)
Inflamación , Ubiquitina , Humanos , Muerte Celular , Membrana Celular , Enzimas Desubicuitinizantes , Inflamación/genética , Síndrome , Complejos de Ubiquitina-Proteína LigasaRESUMEN
Yuri Shibata and David Komander discuss the composition, regulation and functions of the linear ubiquitin chain assembly complex (LUBAC).
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , FN-kappa B/genética , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Work toward rubella elimination has accelerated globally. A reliable laboratory confirmation of rubella-suspected cases is required for effective surveillance in the rubella-elimination phase. The use of adequate specimens is a key to improving the quality of this surveillance. STUDY DESIGN: We conducted rubella virus (RUBV) isolation and RUBV genome or anti-RUBV IgM detection on 1023 specimens from 372 rubella- or measles-suspected cases collected through the national surveillance program in Sakai city of Osaka prefecture, Japan between 2011 and 2013. The resulting data were analyzed by specimen type, collection date, and immunological status. RESULTS: Among the three specimen types (throat swab, serum or plasma, and urine) collected through 10 days post-rash onset, the highest success rates for RUBV genome detection and RUBV isolation were obtained using throat swabs. In agreement with previous work, RUBV-specific IgM were undetectable in 50% of the rubella-confirmed cases until 3 days after rash onset. The success rates of RUBV genome detection and RUBV isolation declined in association with the appearance of RUBV-specific antibodies in blood, especially in serum, plasma, or urine samples. CONCLUSION: Throat swabs are the most optimal specimen types for both RUBV genome detection and RUBV isolation; serum/plasma samples may be suboptimal, especially for RUBV isolation. The findings from this study will provide useful information for improving laboratory surveillance for rubella in the elimination phase.
Asunto(s)
Anticuerpos Antivirales/sangre , Técnicas de Diagnóstico Molecular/normas , Virus de la Rubéola/genética , Rubéola (Sarampión Alemán)/diagnóstico , Pruebas Serológicas/normas , Adolescente , Adulto , Animales , Niño , Preescolar , Chlorocebus aethiops , Femenino , Genoma Viral , Genotipo , Humanos , Inmunoglobulina M/sangre , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Rubéola (Sarampión Alemán)/inmunología , Rubéola (Sarampión Alemán)/virología , Virus de la Rubéola/inmunología , Virus de la Rubéola/aislamiento & purificación , Pruebas Serológicas/métodos , Células Vero , Adulto JovenRESUMEN
Despite various inhibitors targeting the zinc center(s) of enzymes, drugs that target zinc fingers have not been examined in detail. We previously developed a dithiol compound named SN-1 that has an inhibitory effect on the function of zinc finger transcription factors, but its mechanism of action has not yet been elucidated. To establish a general principle for new drugs, the details of the action of SN-1 against a zinc finger protein were examined. As a zinc-finger-containing protein, we focused on TRAF6, which is related to cancer and inflammation. Binding of SN-1 to TRAF6 and its effect on TRAF6 ubiquitination were examined inâ vitro, and the binding mode was calculated by computational methodology. Furthermore, ubiquitination of TRAF6 and downstream signaling was examined by cell-based experiments. The results show that SN-1 binds to TRAF6, inhibiting its auto-ubiquitination and downstream NF-κB signaling. Docking studies indicate that SN-1 binds directly to the first zinc finger of TRAF6. This binding disrupts the neighboring structure, that is, the RING finger domain, to suppress the ubiquitin ligase activity of TRAF6. Taken together, this study provides a platform for developing new small molecules that target zinc finger proteins.
Asunto(s)
Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Tolueno/análogos & derivados , Sitios de Unión/efectos de los fármacos , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Simulación del Acoplamiento Molecular , Estructura Molecular , Factor 6 Asociado a Receptor de TNF/química , Factor 6 Asociado a Receptor de TNF/metabolismo , Tolueno/química , Tolueno/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
The tumor suppressor CYLD belongs to a ubiquitin (Ub)-specific protease (USP) family and specifically cleaves Met1- and Lys63-linked polyubiquitin chains to suppress inflammatory signaling pathways. Here, we report crystal structures representing the catalytic states of zebrafish CYLD for Met1- and Lys63-linked Ub chains and two distinct precatalytic states for Met1-linked chains. In both catalytic states, the distal Ub is bound to CYLD in a similar manner, and the scissile bond is located close to the catalytic residue, whereas the proximal Ub is bound in a manner specific to Met1- or Lys63-linked chains. Further structure-based mutagenesis experiments support the mechanism by which CYLD specifically cleaves both Met1- and Lys63-linked chains and provide insight into tumor-associated mutations of CYLD. This study provides new structural insight into the mechanisms by which USP family deubiquitinating enzymes recognize and cleave Ub chains with specific linkage types.
Asunto(s)
Proteínas Supresoras de Tumor/química , Proteasas Ubiquitina-Específicas/química , Proteínas de Pez Cebra/química , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/química , Peptidasa Específica de Ubiquitina 7 , Proteasas Ubiquitina-Específicas/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The persistent or excess activation of NF-κB causes various inflammatory and autoimmune diseases, but the molecular mechanisms that negatively regulate NF-κB activation are not fully understood. Here we show that p47, an essential factor for Golgi membrane fusion, associates with the NEMO subunit of the IκB kinase (IKK) complex upon TNF-α or IL-1 stimulation, and inhibits IKK activation. p47 binds to Lys63-linked and linear polyubiquitin chains, which are conjugated to NEMO upon such stimulation. The binding of p47 to polyubiquitinated NEMO triggers the lysosomal degradation of NEMO, thereby inhibiting IKK activation. The silencing of p47 results in enhanced TNF-α- or IL-1-induced IKK activation, and an increased expression of genes encoding inflammatory mediators. Taken together, our results suggest that p47 is critical for negatively regulating stimulation-induced IKK activation in a manner that is mechanistically distinct from the previously characterized negative regulators, such as A20 and CYLD.
Asunto(s)
Quinasa I-kappa B/metabolismo , Lisosomas/metabolismo , Poliubiquitina/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Quinasa I-kappa B/genética , Inmunoprecipitación , Células Jurkat/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Sf9 , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genéticaRESUMEN
Activation of NF-κB by human T cell leukaemia virus type 1 Tax is thought to be crucial in T-cell transformation and the onset of adult T cell leukaemia. Tax activates NF-κB through activation of the IκB kinase (IKK) complex, similar to cytokine-induced NF-κB activation, which involves active signalling complex formation using polyubiquitin chains as a platform. Although polyubiquitination of Tax was reported to be required for IKK activation, most studies have been performed using intact cells, in which secondary NF-κB activation can be induced by various cytokines that are secreted due to Tax-mediated primary NF-κB activation. Therefore, a cell-free assay system, in which IKK can be activated by adding highly purified recombinant Tax to cytosolic extract, was used to analyse Tax-induced IKK activation. In contrast to the cytosolic extract, the purified IKK complex was not activated by Tax, whereas, it was efficiently activated by MEKK1, that does not require polyubiquitination to activate IKK. Moreover, Tax-induced IKK activation was blocked when the cytosolic extract was mixed with either lysine-free, methylated or K63R ubiquitin. These results obtained through our cell-free assay suggest that K63-linked polyubiquitination is critical, but linear polyubiquitination is dispensable or insufficient for Tax-induced IKK activation.