RESUMEN
Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Autofagia , Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Relacionadas con la Autofagia , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Proteínas HSP70 de Choque Térmico/metabolismo , Proteína Huntingtina/metabolismo , Masculino , Ratones , Enfermedades Neurodegenerativas/patología , Agregado de Proteínas , Pliegue de Proteína , ProteolisisRESUMEN
HOIL-1, a component of the linear ubiquitin chain assembly complex (LUBAC), ubiquitylates serine and threonine residues in proteins by esterification. Here, we report that mice expressing an E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, heart and other organs, indicating that HOIL-1's E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and α1:4-linked maltoheptaose in vitro and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The monoubiquitylation of maltoheptaose was accelerated > 100-fold by the interaction of Met1-linked or Lys63-linked ubiquitin oligomers with the RBR domain of HOIL-1. HOIL-1 also transferred pre-formed ubiquitin oligomers to maltoheptaose en bloc, producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to unbranched and infrequently branched glucose polymers in vitro, but not to highly branched mammalian glycogen, suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal from cells, preventing precipitation as polyglucosan.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Glucanos , Glucosa , Glucógeno/metabolismo , Mamíferos , Ratones , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The atypical protein kinase ALPK1 is activated by the bacterial nucleotide sugar ADP-heptose and phosphorylates TIFA to switch on a signaling pathway that combats microbial infection. In contrast, ALPK1 mutations cause two human diseases: the ALPK1[T237M] and ALPK1[Y254C] mutations underlie ROSAH syndrome (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis, and migraine headache), while the ALPK1[V1092A] mutation accounts for 45% of spiradenoma and 30% of spiradenocarcinoma cases studied. In this study, we demonstrate that unlike wild-type (WT) ALPK1, the disease-causing ALPK1 mutants trigger the TIFA-dependent activation of an NF-κB/activator protein 1 reporter gene in the absence of ADP-heptose, which can be suppressed by either of two additional mutations in the ADP-heptose binding site that prevent the activation of WT ALPK1 by ADP-heptose. These observations are explained by our key finding that although ALPK1[T237M] and ALPK1[V1092A] are activated by bacterial ADP-heptose, they can also be activated by nucleotide sugars present in human cells (UDP-mannose, ADP-ribose, and cyclic ADP-ribose) which can be prevented by disruption of the ADP-heptose binding site. The ALPK1[V1092A] mutant was also activated by GDP-mannose, which did not activate ALPK1[T237M]. These are new examples of disease-causing mutations permitting the allosteric activation of an enzyme by endogenous molecules that the WT enzyme does not respond to. We propose that the loss of the specificity of ALPK1 for bacterial ADP-heptose underlies ROSAH syndrome and spiradenoma/spiradenocarcinoma caused by ALPK1 mutation.
Asunto(s)
Acrospiroma , Neoplasias de las Glándulas Sudoríparas , Humanos , Nucleótidos/genética , Azúcares , Esplenomegalia , Manosa , Heptosas/metabolismoRESUMEN
Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biocatálisis , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Cisteína/metabolismo , Esterificación , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Moleculares , Dominios Proteicos , Proteómica , Serina/metabolismo , Especificidad por Sustrato , Treonina/metabolismo , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.
Asunto(s)
Enzimas Desubicuitinizantes/genética , Esterasas/genética , Enfermedad de Machado-Joseph/genética , Ubiquitina/genética , Aminoácidos/genética , Enzimas Desubicuitinizantes/aislamiento & purificación , Humanos , Lisina/genética , Enfermedad de Machado-Joseph/enzimología , Enfermedad de Machado-Joseph/patología , Espectrometría de Masas , Procesamiento Proteico-Postraduccional/genética , Ubiquitinación/genéticaRESUMEN
MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration.
Asunto(s)
Axones/metabolismo , Cisteína/metabolismo , Dominios RING Finger , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Femenino , Técnicas de Sustitución del Gen , Humanos , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL/embriología , Ratones Transgénicos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
The concerted action of many protein kinases helps orchestrate the error-free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin-dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α-binding-deficient FAM83DF283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D-/- cells, or artificially delivering CK1α to the spindle in FAM83DF283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Huso Acromático/metabolismo , Animales , Quinasa de la Caseína I/genética , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Citometría de Flujo , Células HeLa , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Mitosis/fisiologíaRESUMEN
Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1-5 (DCNL1-5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain-containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client-substrate ubiquitylation at multiple levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteína NEDD8/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Portadoras/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína NEDD8/genética , Dominios Proteicos , Proteínas , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Our previous studies of PAWS1 (protein associated with SMAD1; also known as FAM83G) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1-knockout U2OS osteosarcoma cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration. Genetic ablation of CD2AP from U2OS cells instigates actin and cell migration defects reminiscent of those seen in PAWS1-knockout cells.This article has an associated First Person interview with the first authors of the paper.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proteínas del Citoesqueleto/genética , Adhesiones Focales/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Transducción de SeñalRESUMEN
The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co-localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.
Asunto(s)
Caseína Quinasa Ialfa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Vía de Señalización Wnt , Animales , Proteína Axina/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular Tumoral , Núcleo Celular , Expresión Génica Ectópica , Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , beta Catenina/metabolismoRESUMEN
Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND-cullin-DCNL complexes might represent 'reserve' CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Oncogénicas/metabolismo , Péptido Sintasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismo , Línea Celular , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína NEDD8 , Unión Proteica , Proteínas , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Mutación , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7â¼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
Asunto(s)
Proteínas Portadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Células HEK293 , Humanos , Proteína NEDD8 , Pirimidinas/farmacología , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Ubiquitinas/antagonistas & inhibidores , Ubiquitinas/metabolismoRESUMEN
In November 2015 it became apparent that a person with dementia's journey through the acute hospital was not always as streamlined as it should have been. There was evidence of late and multiple inter-ward transfers for this patient group that could potentially have a detrimental effect on individuals' and carers' well-being. The aim of this project was to examine current processes around patient flow and decision-making, explore any themes arising and identify opportunities for improving transitions of care. Collaborative working among various specialties has resulted in increased transfers before 8pm, a reduction in transfers after midnight and a reduction in inter-ward transfers.
Asunto(s)
Conducta Cooperativa , Demencia/terapia , Hospitalización , Mejoramiento de la Calidad , Cuidado de Transición/organización & administración , HumanosRESUMEN
BACKGROUND: FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons. RESULTS: We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation. CONCLUSIONS: The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings indicate that the repertoire of VAPB interactors is more diverse than previously anticipated and link VAPB to the function of ATPase complexes such as p97/FAF1 and ASNA1/TRC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Adenosina Trifosfatasas/metabolismo , ATPasas Transportadoras de Arsenitos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/genética , Proteínas Reguladoras de la Apoptosis , ATPasas Transportadoras de Arsenitos/química , Hexosiltransferasas , Humanos , Inmunoprecipitación , Espectrometría de Masas , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteolisis/efectos de los fármacos , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacos , Proteínas de Transporte Vesicular/química , Proteínas de Unión al GTP rab3/metabolismoAsunto(s)
Dispepsia , Esofagitis Eosinofílica , Duodeno , Humanos , Inhibidores de la Bomba de Protones , Bombas de ProtonesRESUMEN
The WNK (with no lysine kinase)-SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin-RING E3 ubiquitin ligase complexes. To explore how a CUL3-KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na(+)/Cl(-) co-transporter)/NKCC1 (Na(+)/K(+)/2Cl(-) co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3-KLHL3 E3 ligase complex, but not a disease-causing CUL3-KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479-667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479-667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3-KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Mutación Missense , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/enzimología , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales , Sustitución de Aminoácidos , Proteínas Portadoras/genética , Proteínas Cullin/genética , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Proteínas Serina-Treonina Quinasas/genética , Seudohipoaldosteronismo/genética , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a 'hemiRING' zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2-E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2â¼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Catálisis , Ubiquitinación , Proteínas de Unión a Calmodulina/metabolismoRESUMEN
Reversible phosphorylation of the transcription factor EB (TFEB) coordinates cellular responses to metabolic and other stresses. During nutrient replete and stressor-free conditions, phosphorylated TFEB is primarily localized to the cytoplasm. Stressor-mediated reduction of TFEB phosphorylation promotes its nuclear translocation and context-dependent transcriptional activity. In this study, we explored targeted dephosphorylation of TFEB as an approach to activate TFEB in the absence of nutrient deprivation or other cellular stress. Through an induction of proximity between TFEB and several phosphatases using the AdPhosphatase system, we demonstrate targeted dephosphorylation of TFEB in cells. Furthermore, by developing a heterobifunctional molecule BDPIC (bromoTAG-dTAG proximity-inducing chimera), we demonstrate targeted dephosphorylation of TFEB-dTAG through induced proximity to bromoTAG-PPP2CA. Targeted dephosphorylation of TFEB-dTAG by bromoTAG-PPP2CA with BDPIC at the endogenous levels is sufficient to induce nuclear translocation and some transcriptional activity of TFEB.
RESUMEN
TGF-ß (transforming growth factor-ß) signaling is involved in a myriad of cellular processes and its dysregulation has been implicated in many human diseases, including fibrosis and cancer. TGF-ß transcriptional responses are controlled by tail phosphorylation of transcription factors SMAD2 and SMAD3 (mothers against decapentaplegic homolog 2/3). Therefore, targeted dephosphorylation of phospho-SMAD3 could provide an innovative mechanism to block some TGF-ß-induced transcriptional responses, such as the transcription of SERPINE-1, which encodes plasminogen activator inhibitor 1 (PAI-1). Here, by developing and employing a bifunctional molecule, BDPIC (bromoTAG-dTAG proximity-inducing chimera), we redirected multiple phosphatases, tagged with bromoTAG, to dephosphorylate phospho-SMAD3, tagged with dTAG. Using CRISPR-Cas9 technology, we generated homozygous double knock-in A549 bromoTAG/bromoTAG PPM1H/ dTAG/dTAG SMAD3 cells, in which the BDPIC-induced proximity between bromoTAG-PPM1H and dTAG-SMAD3 led to a robust dephosphorylation of dTAG-SMAD3 and a significant decrease in SERPINE-1 transcription. Our work demonstrates targeted dephosphorylation of phospho-proteins as an exciting modality for rewiring cell signaling.