RESUMO
Oculocutaneous albinism (OCA) is a genetically heterogeneous group of autosomal recessive disorders, which presents with decreased or absent pigmentation in the hair, skin, and eyes. OCA1, as a subtype of OCA, is caused by mutations in the tyrosinase gene (TYR). In this study, we performed in vitro functional analysis of eight TYR variants (one frameshift variant: c.929dupC (p.Arg311Lysfs*7); seven missense variants: c.896G>A (p.Arg299His), c.1234C>A (p.Pro412Thr), c.1169A>G (p.His390Arg), c.937C>A (p.Pro313Thr), c.636A>T (p.Arg212Ser), c.623 T>G (p.Leu208Arg), c.1325C>A (p.Ser442Tyr)) identified in Chinese OCA families. TYR plasmids were transfected into HEK 293 T cells to explore the effects of TYR variants on their processing, protein expression, activity, and degradation. The results showed that all eight variants caused TYR to be retained in the endoplasmic reticulum (ER), processing was blocked, and TYR activity almost disappeared; the frameshift variant caused the size of the TYR protein to be reduced by about 30KD, and the protein expression of the remaining seven missense variants was reduced; the ER-associated degradation (ERAD) pathway mediates the degradation of TYR variants that occur on the Tyrosinase copper-binding domain, while the degradation of TYR variants that are not located on that domain may be mediated by a new degradation pathway--ER-to-lysosome-associated degradation (ERLAD). In summary, TYR variants affected their protein processing and activity, and may also induce ER stress and trigger degradation through the ERLAD pathway in addition to the ERAD degradation pathway, providing new insights into the potential pathogenic mechanism for OCA1 caused by TYR variants.
Assuntos
Albinismo Oculocutâneo , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Monofenol Mono-Oxigenase , Humanos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Células HEK293 , Albinismo Oculocutâneo/genética , Albinismo Oculocutâneo/metabolismo , Retículo Endoplasmático/metabolismo , Mutação de Sentido Incorreto , Povo Asiático/genética , Feminino , Masculino , Proteólise , Linhagem , População do Leste AsiáticoRESUMO
Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.
Assuntos
Apresentação de Antígeno , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Antígenos de Histocompatibilidade Classe I , Proteínas de Membrana Transportadoras , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of diet-induced obesity (DIO) and type 2 diabetes; however, the underlying molecular mechanism remain vague and debatable. Here we report that SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC-expressing neurons ameliorates diet-induced obesity and its associated complications, partly by regulating the turnover of the long isoform of Leptin receptors (LepRb). Loss of SEL1L in POMC-expressing neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including fatty liver, glucose intolerance, insulin and leptin resistance. Mechanistically, nascent LepRb, both wildtype and disease-associated Cys604Ser variant, are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. In the absence of SEL1L-HRD1 ERAD, LepRb are largely retained in the ER, in an ER stress-independent manner. This study uncovers an important role of SEL1L-HRD1 ERAD in the pathogenesis of central leptin resistance and leptin signaling.
Assuntos
Dieta Hiperlipídica , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Leptina , Neurônios , Obesidade , Pró-Opiomelanocortina , Receptores para Leptina , Transdução de Sinais , Ubiquitina-Proteína Ligases , Animais , Obesidade/metabolismo , Obesidade/genética , Obesidade/etiologia , Obesidade/patologia , Neurônios/metabolismo , Leptina/metabolismo , Receptores para Leptina/metabolismo , Receptores para Leptina/genética , Camundongos , Pró-Opiomelanocortina/metabolismo , Pró-Opiomelanocortina/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Dieta Hiperlipídica/efeitos adversos , Masculino , Retículo Endoplasmático/metabolismo , Hipotálamo/metabolismo , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Proteínas/metabolismo , Proteínas/genética , Camundongos Knockout , Humanos , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Cone photoreceptor cyclic nucleotide-gated (CNG) channels play an essential role in phototransduction and cellular Ca2+ homeostasis. Mutations in genes encoding the channel subunits CNGA3 and CNGB3 are associated with achromatopsia, progressive cone dystrophy, and early-onset macular degeneration. Cone loss in patients with achromatopsia and cone dystrophy associated with CNG channel mutations has been documented by optical coherence tomography and in mouse models of CNG channel deficiency. Cone death in CNG channel-deficient retinas involves endoplasmic reticulum (ER) stress-associated apoptosis, dysregulation of cellular/ER Ca2+ homeostasis, impaired protein folding/processing, and impaired ER-associated degradation (ERAD). The E3 ubiquitin-protein ligase synoviolin 1 (SYVN1) is the primary component of the SYVN1/SEL1L ER retrotranslocon responsible for ERAD. Previous studies have shown that manipulations that protect cones and reduce ER stress/cone death in CNG channel deficiency, such as increasing ER Ca2+ preservation or treatment with an ER chaperone, increase the expression of SYVN1 and other components of the ER retrotranslocon. The present work investigated the effects of SYVN1 overexpression. Intraocular injection of AAV5-IRBP/GNAT2-Syvn1 resulted in overexpression of SYVN1 in cones of CNG channel-deficient mice. Following treatment, cone density in Cnga3-/- mice was significantly increased, compared with untreated controls, outer segment localization of cone opsin was improved, and ER stress/apoptotic cell death was reduced. Overexpression of SYVN1 also led to increased expression levels of the retrotranslocon components, degradation in ER protein 1 (DERL1), ERAD E3 ligase adaptor subunit (SEL1L), and homocysteine inducible ER protein with ubiquitin-like domain 1 (HERPUD1). Moreover, overexpression of SYVN1 likely enhanced protein ubiquitination/proteasome degradation in CNG channel-deficient retinas. This study demonstrates the role of SYVN1/ERAD in cone preservation in CNG channel deficiency and supports the strategy of promoting ERAD for cone protection.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Células Fotorreceptoras Retinianas Cones , Ubiquitina-Proteína Ligases , Animais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Camundongos , Retículo Endoplasmático/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Degradação Associada com o Retículo Endoplasmático , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
As the largest organelle in eukaryotic cells, the endoplasmic reticulum (ER) plays a crucial role in regulating intracellular protein folding, translation and assembly. Multiple quality control mechanisms in the ER ensure accurate modification of proteins in the ER lumen are accurately modified, thus maintaining calcium homeostasis, oxidative stress, cellular senescence and apoptosis. These mechanisms include ER stress (ERS), ER autophagy (ER-phagy, ERPA) and ER-associated degradation (ERAD). Intervertebral disc degeneration (IDD) is an age-related degenerative disease of the spine. Although the pathogenesis of IDD has not been fully elucidated, emerging evidence suggests that the ER quality control system may be involved in its progression. Previous studies have focused on mitochondrial quality control and its related mechanisms in diseases, with limited systematic summaries on the ER quality control system. In this paper, we comprehensively reviewed the molecular mechanisms of the ER quality control system and investigated its association with IDD. In addition, we summarized the potential therapeutic strategies targeting the ER quality control system to attenuate IDD progression, offering new insights into the pathogenesis and regenerative repair strategies of IDD.
Assuntos
Autofagia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Degeneração do Disco Intervertebral , Humanos , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/fisiopatologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Autofagia/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Animais , Degradação Associada com o Retículo Endoplasmático/fisiologiaRESUMO
Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.
Assuntos
Cistationina beta-Sintase , Homocistinúria , Mutação de Sentido Incorreto , Proteólise , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/química , Humanos , Homocistinúria/genética , Homocistinúria/metabolismo , Células HEK293 , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitinação , Degradação Associada com o Retículo EndoplasmáticoRESUMO
Endoplasmic reticulum (ER)-associated degradation (ERAD) plays key roles in controlling protein levels and quality in eukaryotes. The Ring Finger Protein 185 (RNF185)/membralin ubiquitin ligase complex was recently identified as a branch in mammals and is essential for neuronal function, but its function in plant development is unknown. Here, we report the map-based cloning and characterization of Narrow Leaf and Dwarfism 1 (NLD1), which encodes the ER membrane-localized protein membralin and specifically interacts with maize homologs of RNF185 and related components. The nld1 mutant shows defective leaf and root development due to reduced cell number. The defects of nld1 were largely restored by expressing membralin genes from Arabidopsis thaliana and mice, highlighting the conserved roles of membralin proteins in animals and plants. The excessive accumulation of ß-hydroxy ß-methylglutaryl-CoA reductase in nld1 indicates that the enzyme is a membralin-mediated ERAD target. The activation of bZIP60 mRNA splicing-related unfolded protein response signaling and marker gene expression in nld1, as well as DNA fragment and cell viability assays, indicate that membralin deficiency induces ER stress and cell death in maize, thereby affecting organogenesis. Our findings uncover the conserved, indispensable role of the membralin-mediated branch of the ERAD pathway in plants. In addition, ZmNLD1 contributes to plant architecture in a dose-dependent manner, which can serve as a potential target for genetic engineering to shape ideal plant architecture, thereby enhancing high-density maize yields.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteínas de Plantas , Ubiquitina-Proteína Ligases , Zea mays , Zea mays/genética , Zea mays/metabolismo , Zea mays/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Retículo Endoplasmático/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica de Plantas , Estresse do Retículo Endoplasmático , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Folhas de Planta/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Resposta a Proteínas não DobradasRESUMO
The endoplasmic reticulum (ER) employs stringent quality control mechanisms to ensure the integrity of protein folding, allowing only properly folded, processed and assembled proteins to exit the ER and reach their functional destinations. Mutant proteins unable to attain their correct tertiary conformation or form complexes with their partners are retained in the ER and subsequently degraded through ER-associated protein degradation (ERAD) and associated mechanisms. ER retention contributes to a spectrum of monogenic diseases with diverse modes of inheritance and molecular mechanisms. In autosomal dominant diseases, when mutant proteins get retained in the ER, they can interact with their wild-type counterparts. This interaction may lead to the formation of mixed dimers or aberrant complexes, disrupting their normal trafficking and function in a dominant-negative manner. The combination of ER retention and dominant-negative effects has been frequently documented to cause a significant loss of functional proteins, thereby exacerbating disease severity. This review aims to examine existing literature and provide insights into the impact of dominant-negative effects exerted by mutant proteins retained in the ER in a range of autosomal dominant diseases including skeletal and connective tissue disorders, vascular disorders, neurological disorders, eye disorders and serpinopathies. Most crucially, we aim to emphasize the importance of this area of research, offering substantial potential for understanding the factors influencing phenotypic variability associated with genetic variants. Furthermore, we highlight current and prospective therapeutic approaches targeted at ameliorating the effects of mutations exhibiting dominant-negative effects. These approaches encompass experimental studies exploring treatments and their translation into clinical practice.
Assuntos
Retículo Endoplasmático , Humanos , Retículo Endoplasmático/metabolismo , Genes Dominantes , Degradação Associada com o Retículo Endoplasmático , Dobramento de Proteína , MutaçãoRESUMO
RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.
Assuntos
Besouros , Citosol , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , RNA de Cadeia Dupla , Proteínas de Ligação a RNA , Animais , Retículo Endoplasmático/metabolismo , RNA de Cadeia Dupla/metabolismo , Citosol/metabolismo , Besouros/metabolismo , Besouros/genética , Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Interferência de RNA , Transporte BiológicoRESUMO
Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic ß-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic ß-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of ß-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.
Assuntos
Autoantígenos , Degradação Associada com o Retículo Endoplasmático , Proinsulina , Proteólise , Enzimas de Conjugação de Ubiquitina , Humanos , Apresentação de Antígeno/imunologia , Autoantígenos/metabolismo , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/imunologia , Proinsulina/metabolismo , Proinsulina/imunologia , Proinsulina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.
Assuntos
Vírus da Encefalite Japonesa (Espécie) , Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana , Ubiquitina-Proteína Ligases , Replicação Viral , Humanos , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Vírus da Encefalite Japonesa (Espécie)/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células HeLa , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Interações Hospedeiro-Patógeno , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Proteínas/metabolismo , Proteínas/genética , Antígenos de DiferenciaçãoRESUMO
A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.
Assuntos
Retículo Endoplasmático , Epilepsias Mioclônicas , Proteólise , Receptores de GABA-A , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Animais , Retículo Endoplasmático/metabolismo , Camundongos , Humanos , Convulsões Febris/metabolismo , Convulsões Febris/genética , Degradação Associada com o Retículo Endoplasmático , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Mutação , Células HEK293 , Chaperona BiP do Retículo Endoplasmático/metabolismoRESUMO
The endoplasmic reticulum (ER) is important to cells because of its essential functions, including synthesizing three major nutrients and ion transport. When cellular homeostasis is disrupted, ER quality control (ERQC) system is activated effectively to remove misfolded and unfolded proteins through ER-phagy, ER-related degradation (ERAD), and molecular chaperones. When unfolded protein response (UPR) and ER stress are activated, the cell may be suffering a huge blow, and the most probable consequence is apoptosis. The membrane contact points between the ER and sub-organelles contribute to communication between the organelles. The decrease in oxygen concentration affects the morphology and structure of the ER, thereby affecting its function and further disrupting the stable state of cells, leading to the occurrence of disease. In this study, we describe the functions of ER-, ERQC-, and ER-related membrane contact points and their changes under hypoxia, which will help us further understand ER and treat ER-related diseases.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Humanos , Animais , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Hipóxia/metabolismo , Apoptose/fisiologia , Hipóxia Celular/fisiologia , Degradação Associada com o Retículo EndoplasmáticoRESUMO
The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Endorribonucleases , Leucemia de Mastócitos , Proteínas Serina-Treonina Quinases , Humanos , Linhagem Celular Tumoral , Degradação Associada com o Retículo Endoplasmático/genética , Endorribonucleases/metabolismo , Leucemia de Mastócitos/metabolismo , Leucemia de Mastócitos/patologia , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína com Valosina/metabolismo , Proteína com Valosina/genéticaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia worldwide. Due to its uncertain pathogenesis, there is currently no treatment available for AD. Increasing evidences have linked cellular senescence to AD, although the mechanism triggering cellular senescence in AD requires further exploration. To investigate the involvement of cellular senescence in AD, we explored the effects of cadmium chloride (CdCl2) exposure, one of the potential environmental risk factors for AD, on neuron senescence in vivo and in vitro. ß-amyloid (Aß) and tubulin-associated protein (tau) pathologies were found to be enhanced by CdCl2 exposure in the in vitro models, while p53/p21/Rb cascade-related neuronal senescence pathways were activated. Conversely, the use of melatonin, a cellular senescence inhibitor, or a cadmium ion chelator suppressed CdCl2-induced neuron senescence, along with the Aß and tau pathologies. Mechanistically, CdCl2 exposure activated the suppressor enhancer Lin-12/Notch 1-like (SEL1L)/HMG-CoA reductase degradation 1 (HRD1)-regulated endoplasmic reticulum-associated degradation (ERAD), which enhanced the ubiquitin degradation of sigma-1 receptor (SigmaR1) by specifically recognizing its K142 site, resulting in the activation of the p53/p21/Rb pathway via the induction of Ca2+ dyshomeostasis and mitochondrial dysfunction. In the in vivo models, the administration of the SigmaR1 agonist ANAVEX2-73 rescues neurobehavioral inhibition and alleviates cellular senescence and AD-like pathology in the brain tissue of CdCl2-exposed mice. Consequently, the present study revealed a novel senescence-associated regulatory route for the SEL1L/HRD1/SigmaR1 axis that affects the pathological progression of CdCl2 exposure-associated AD. CdCl2 exposure activated SEL1L/HRD1-mediated ERAD and promoted the ubiquitinated degradation of SigmaR1, activating p53/p21/Rb pathway-regulated neuronal senescence. The results of the present study suggest that SigmaR1 may function as a neuroprotective biomarker of neuronal senescence, and pharmacological activation of SigmaR1 could be a promising intervention strategy for AD therapy.
Assuntos
Cloreto de Cádmio , Senescência Celular , Degradação Associada com o Retículo Endoplasmático , Neurônios , Receptores sigma , Animais , Senescência Celular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cloreto de Cádmio/toxicidade , Receptores sigma/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Camundongos , Proteínas tau/metabolismo , Masculino , Doença de Alzheimer/metabolismo , Humanos , Melatonina/farmacologia , Camundongos Endogâmicos C57BLRESUMO
The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Lisossomos , Proteínas de Membrana , Lisossomos/metabolismo , Humanos , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Secretases da Proteína Precursora do Amiloide/metabolismo , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/genética , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dobramento de Proteína , Transporte Proteico , Lectinas/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Camundongos , Células HeLaRESUMO
Mutations in NKCC2 generate antenatal Bartter syndrome type 1 (type 1 BS), a life-threatening salt-losing nephropathy characterized by arterial hypotension, as well as electrolyte abnormalities. In contrast to the genetic inactivation of NKCC2, inappropriate increased NKCC2 activity has been associated with salt-sensitive hypertension. Given the importance of NKCC2 in salt-sensitive hypertension and the pathophysiology of prenatal BS, studying the molecular regulation of this Na-K-2Cl cotransporter has attracted great interest. Therefore, several studies have addressed various aspects of NKCC2 regulation, such as phosphorylation and post-Golgi trafficking. However, the regulation of this cotransporter at the pre-Golgi level remained unknown for years. Similar to several transmembrane proteins, export from the ER appears to be the rate-limiting step in the cotransporter's maturation and trafficking to the plasma membrane. The most compelling evidence comes from patients with type 5 BS, the most severe form of prenatal BS, in whom NKCC2 is not detectable in the apical membrane of thick ascending limb (TAL) cells due to ER retention and ER-associated degradation (ERAD) mechanisms. In addition, type 1 BS is one of the diseases linked to ERAD pathways. In recent years, several molecular determinants of NKCC2 export from the ER and protein quality control have been identified. The aim of this review is therefore to summarize recent data regarding the protein quality control of NKCC2 and to discuss their potential implications in BS and blood pressure regulation.
Assuntos
Síndrome de Bartter , Pressão Sanguínea , Membro 1 da Família 12 de Carreador de Soluto , Síndrome de Bartter/metabolismo , Síndrome de Bartter/genética , Humanos , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Degradação Associada com o Retículo EndoplasmáticoRESUMO
Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Proteínas de Membrana , Ubiquitina-Proteína Ligases , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Ligação a DNA , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.
Assuntos
Linfócitos T CD8-Positivos , Estresse do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Memória Imunológica , Animais , Humanos , Camundongos , Doença Aguda , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Coriomeningite Linfocítica/patologia , Camundongos Endogâmicos C57BL , Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Masculino , FemininoRESUMO
ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.