RESUMO
Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.
Assuntos
Ferroptose , Células-Tronco Hematopoéticas , Humanos , Endopeptidases/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.
Assuntos
Infecções por Coronavirus , Coronavirus , Dipeptidil Peptidase 4 , Pangolins , Animais , Humanos , Camundongos , Quirópteros , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Endopeptidases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeo Hidrolases/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Coronavirus/fisiologiaRESUMO
We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.
Assuntos
Antígenos CD28/deficiência , Padrões de Herança/genética , Papillomaviridae/fisiologia , Pele/virologia , Linfócitos T/imunologia , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Criança , Endopeptidases/metabolismo , Feminino , Genes Recessivos , Células HEK293 , Homozigoto , Humanos , Imunidade Humoral , Memória Imunológica , Células Jurkat , Queratinócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Oncogenes , Papiloma/patologia , Papiloma/virologia , Linhagem , Sinais Direcionadores de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Endopeptidases/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aß peptides. The shift in Aß length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aß peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aß. In contrast, E-Aßn stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aß production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Presenilina-1/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Endopeptidases , Estabilidade Enzimática , Feminino , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Mutação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Presenilina-1/química , Presenilina-1/genéticaRESUMO
Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.
Assuntos
Doenças Autoimunes/genética , Autoimunidade/genética , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Inflamação/genética , Animais , Anticorpos Neutralizantes/uso terapêutico , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Linfócitos B/imunologia , Citocinas/metabolismo , Enzimas Desubiquitinantes/genética , Modelos Animais de Doenças , Endopeptidases/genética , Mutação em Linhagem Germinativa , Humanos , Inflamação/imunologia , Inflamação/terapia , Infliximab/uso terapêutico , Metionina/metabolismo , Camundongos , Camundongos Mutantes , Células Mieloides/imunologia , Poliubiquitina/metabolismo , Deleção de Sequência , Síndrome , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
In this issue, Liu et al. present an in-depth study aiming to unravel the structural, biochemical, and physiological aspects of how type III-E CRISPR-Cas systems trigger abortive infection by activating a protease upon target RNA recognition.1.
Assuntos
Sistemas CRISPR-Cas , Endopeptidases , Peptídeo Hidrolases , RNA , BiologiaRESUMO
The acquisition of a protective vertebrate immune system hinges on the efficient generation of a diverse but self-tolerant repertoire of T cells by the thymus through mechanisms that remain incompletely resolved. Here we identified the endosomal-sorting-complex-required-for-transport (ESCRT) protein CHMP5, known to be required for the formation of multivesicular bodies, as a key sensor of thresholds for signaling via the T cell antigen receptor (TCR) that was essential for T cell development. CHMP5 enabled positive selection by promoting post-selection thymocyte survival in part through stabilization of the pro-survival protein Bcl-2. Accordingly, loss of CHMP5 in thymocyte precursor cells abolished T cell development, a phenotype that was 'rescued' by genetic deletion of the pro-apoptotic protein Bim or transgenic expression of Bcl-2. Mechanistically, positive selection resulted in the stabilization of CHMP5 by inducing its interaction with the deubiquitinase USP8. Our results thus identify CHMP5 as an essential component of the post-translational machinery required for T cell development.
Assuntos
Diferenciação Celular/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Proteína 11 Semelhante a Bcl-2/imunologia , Endopeptidases/imunologia , Immunoblotting , Imunoprecipitação , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timócitos/citologia , Ubiquitina Tiolesterase/imunologiaRESUMO
Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidases/metabolismo , Dobramento de Proteína , Proteolipídeos/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína com ValosinaRESUMO
OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.
Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Endopeptidases/genética , Complexos Multiproteicos/metabolismo , Neovascularização Patológica/genética , Poliubiquitina/metabolismo , Adulto , Animais , Endopeptidases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Fator 2 de Diferenciação de Crescimento/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Mutantes , Mutação , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Telangiectasia Hemorrágica Hereditária , Ubiquitina-Proteína Ligases/metabolismoRESUMO
During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.
Assuntos
Endopeptidases , Pseudomonas aeruginosa , Pseudomonas aeruginosa/metabolismo , Endopeptidases/metabolismo , ProteóliseRESUMO
The modification of eukaryotic proteins by isoprenoid lipids, which is known as prenylation, controls the localization and activity of a range of proteins that have crucial functions in biological regulation. The roles of prenylated proteins in cells are well conserved across species, underscoring the biological and evolutionary importance of this lipid modification pathway. Genetic suppression and pharmacological inhibition of the protein prenylation machinery have provided insights into several cellular processes and into the aetiology of diseases in which prenylation is involved. The functional dependence of prenylation substrates, such as RAS proteins, on this modification and the therapeutic potential of targeting the prenylation process in pathological conditions accentuate the need to fully understand this form of post-translational modification.
Assuntos
Alquil e Aril Transferases/metabolismo , Prenilação de Proteína , Processamento de Proteína Pós-Traducional , Terpenos/metabolismo , Proteínas ras/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/genética , Animais , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Endopeptidases/genética , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Neoplasias/prevenção & controle , Transporte Proteico , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
Linear ubiquitin (Ub) plays a role in nuclear factor κB signaling, but the deubiquitinating enzyme that disassembles these chains was unknown. In this issue of Cell, Keusekotten et al. identify a new enzyme that disassembles linear chains with the use of a mechanism that relies on Ub itself to help catalyze peptide bond cleavage.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Animais , HumanosRESUMO
The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Cristalografia por Raios X , Citocinas/metabolismo , Endopeptidases/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Poliubiquitina/biossíntese , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transdução de SinaisRESUMO
ER-associated degradation clears the secretory pathway of misfolded proteins and mediates the regulated degradation of some ER resident proteins. Only a minor increase in the interaction between a protein and a ubiquitin ligase is sufficient to signal substrate degradation. Zhang et al. have identified deubiquitination as a signal amplifier.
Assuntos
Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , HumanosRESUMO
Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1' and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.
Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Neoplasias Ovarianas/enzimologia , Ubiquitinação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Endopeptidases/genética , Feminino , Humanos , Modelos Moleculares , Neoplasias Ovarianas/metabolismo , Estrutura Terciária de Proteína , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Ubiquitinas/metabolismoRESUMO
Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 µM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.
Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteólise , Sequência de Aminoácidos , Bactérias/enzimologia , Membrana Celular/química , Membrana Celular/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Newly synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. The mechanisms that convert modest differences in substrate-ligase interactions into decisive outcomes of ubiquitination are not well understood. Here, we reconstitute membrane protein recognition and ubiquitination in liposomes using purified components from a viral-mediated degradation pathway. We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control.
Assuntos
Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Antígenos CD4/química , Antígenos CD4/metabolismo , Células HEK293 , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins.
Assuntos
Endopeptidases/metabolismo , Proteína NEDD8/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Anticorpos de Domínio Único/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Endopeptidases/imunologia , Células HeLa , Humanos , Proteína NEDD8/imunologia , Proteínas Nucleares/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteólise , Anticorpos de Domínio Único/imunologia , Fatores de Transcrição/imunologia , UbiquitinaçãoRESUMO
N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.