RESUMO
Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While Cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-01 derivatives that target casein kinase 1 alpha (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and a rationale for the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.
RESUMO
The attachment of the ubiquitin-like protein ISG15 to substrates by specific E1-E2-E3 enzymes is a well-established signalling mechanism of the innate immune response. Here, we present a 3.45 Å cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L (also known as UBA7). Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validate the structure and confirm the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enables the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study helps to define the molecular basis of these interactions and the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.
Assuntos
Citocinas , Ubiquitinas , Citocinas/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas ViraisRESUMO
Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation. Given the homology between HOIL-1 and ARIH1, an RBR E3 that functions with the large superfamily of cullin-RING E3 ligases (CRLs), a biochemical investigation was undertaken, showing ARIH1 catalyzes Ser ubiquitylation to CRL-bound substrates. However, the efficiency of ubiquitylation was exquisitely dependent on the location and chemical environment of the Ser residue within the primary structure of the substrate. Comprehensive mutagenesis of the ARIH1 Rcat domain identified residues whose mutation severely impacted both oxyester and isopeptide bond formation at the preferred site for Ser ubiquitylation while only modestly affecting Lys ubiquitylation at the physiological site. The results reveal dual isopeptide and oxyester protein ubiquitylation activities of ARIH1 and set the stage for physiological investigations into this function of emerging importance.
Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Proteínas/metabolismo , CatáliseRESUMO
The cullin-RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs.
Assuntos
Proteínas Culina , Ubiquitina-Proteína Ligases , Proteínas Culina/genética , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteína NEDD8/metabolismoRESUMO
Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of â¼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.
Assuntos
Proteínas Culina , Proteínas F-Box , Proteínas Ligases SKP Culina F-Box , Fatores de Transcrição , Humanos , Proteínas Culina/química , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Conformação Molecular , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.
Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Transporte , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.
RESUMO
Most cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7FBXW8 assembly. CUL7's exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1-FBXW8 to a neddylated CUL1-RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL-CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.
Assuntos
Proteínas Culina , Proteínas F-Box , Proteínas de Transporte/metabolismo , Catálise , Proteínas Culina/química , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Humanos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting.
Assuntos
Proteínas Culina/metabolismo , Proteína NEDD8/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Microscopia Crioeletrônica , Cristalização , Proteínas Culina/genética , Regulação da Expressão Gênica , Humanos , Insetos , Modelos Moleculares , Proteína NEDD8/genética , Conformação Proteica , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores Imunológicos/farmacologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Chaperonas Moleculares/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Células Tumorais CultivadasRESUMO
E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.
Assuntos
Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Regulação Alostérica , Biocatálise , Microscopia Crioeletrônica , Ciclina E/metabolismo , Humanos , Fosforilação , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
RING E3s comprise the largest family of ubiquitin (UB) and ubiquitin-like protein (UBL) ligases. RING E3s typically promote UB or UBL transfer from the active site of an associated E2 enzyme to a distally-recruited substrate. Many RING E3s - including the cullin-RING ligase family - are multifunctional, interacting with various E2s (or other E3s) to target distinct proteins, transfer different UBLs, or to initially modify substrates with UB or subsequently elongate UB chains. Here we consider recent structures of cullin-RING ligases, and their partner E2 enzymes, representing ligation reactions. The studies collectively reveal multimodal mechanisms - interactions between ancillary E2 or E3 domains, post-translational modifications, or auxiliary binding partners - directing cullin-RING E3-E2 enzyme active sites to modify their specific targets.
Assuntos
Proteínas Culina , Ubiquitina , Domínio Catalítico , Proteínas Culina/genética , Proteínas Culina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Imunidade Inata , SARS-CoV-2/enzimologia , SARS-CoV-2/imunologia , Animais , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Citocinas/química , Citocinas/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons/imunologia , Interferons/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , NF-kappa B/imunologia , NF-kappa B/metabolismo , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1ß-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
Assuntos
Microscopia Crioeletrônica , Proteína NEDD8/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Biocatálise , Humanos , Modelos Moleculares , Proteína NEDD8/química , Proteína NEDD8/ultraestrutura , Inibidor de NF-kappaB alfa/química , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/ultraestrutura , Fosforilação , Conformação Proteica , Especificidade por Substrato , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , UbiquitinaçãoRESUMO
The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFß complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.
Assuntos
Desaminase APOBEC-3G/metabolismo , Proteínas Culina/metabolismo , Infecções por HIV/patologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Humanos , Modelos Teóricos , Proteólise , Proteoma/análise , Replicação ViralRESUMO
In eukaryotes, deubiquitinases (DUBs) remove ubiquitin conjugates from diverse substrates, altering their stabilities, localizations or activities. Here we show that many DUBs of the USP and UCH subfamilies can be reversibly inactivated upon oxidation by reactive oxygen species in vitro and in cells. Oxidation occurs preferentially on the catalytic cysteine, abrogating the isopeptide-cleaving activity without affecting these enzymes' affinity to ubiquitin. Sensitivity to oxidative inhibition is associated with DUB activation wherein the active site cysteine is converted to a deprotonated state prone to oxidation. We demonstrate that this redox regulation is essential for mono-ubiquitination of proliferating-cell nuclear antigen in response to oxidative DNA damage, which initiates a DNA damage-tolerance programme. These findings establish a novel mechanism of DUB regulation that may be integrated with other redox-dependent signalling circuits to govern cellular adaptation to oxidative stress, a process intimately linked to aging and cancer.
Assuntos
Endopeptidases/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Animais , Biocatálise/efeitos dos fármacos , Cisteína/metabolismo , Ditiotreitol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Cinética , Camundongos , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Endoplasmic reticulum-associated degradation (ERAD) employs membrane-bound ubiquitin ligases and the translocation-driving ATPase p97 to retrotranslocate misfolded proteins for proteasomal degradation. How retrotranslocated polypeptides bearing exposed hydrophobic motifs or transmembrane domains (TMDs) avoid aggregation before reaching the proteasome is unclear. Here we identify a ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. In vitro, Bag6, the central component of the complex, contains a chaperone-like activity capable of maintaining an aggregation-prone substrate in an unfolded yet soluble state. The physiological importance of this holdase activity is underscored by observations that ERAD substrates accumulate in detergent-insoluble aggregates in cells depleted of Bag6, or of Trc35, a cofactor that keeps Bag6 outside the nucleus for engagement in ERAD. Our results reveal a ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells.
Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Solubilidade , Proteínas Virais/metabolismoRESUMO
Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.