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1.
Trends Biochem Sci ; 2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38906726

RESUMO

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.

2.
Proc Natl Acad Sci U S A ; 121(12): e2309326121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38483986

RESUMO

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.


Assuntos
Proteínas de Choque Térmico HSP70 , Dobramento de Proteína , Humanos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Nucleotídeos , Trifosfato de Adenosina/metabolismo
3.
Development ; 149(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36052695

RESUMO

Stomata are epidermal pores that control gas exchange between plants and the atmosphere. In Arabidopsis, the ERECTA family (ERECTAf) receptors, including ERECTA, ERECTA-LIKE 1 (ERL1) and ERL2, redundantly play pivotal roles in enforcing the 'one-cell-spacing' rule. Accumulating evidence has demonstrated that the functional specificities of receptors are likely associated with their differential subcellular dynamics. The endoplasmic reticulum (ER)-resident chaperone complex SDF2-ERdj3B-BiP functions in many aspects of plant development. We employed pharmacological treatments combined with cell biological and biochemical approaches to demonstrate that the abundance of ERECTA was reduced in the erdj3b-1 mutant, but the localization and dynamics of ERECTA were not noticeably affected. By contrast, the erdj3b mutation caused the retention of ERL1/ERL2 in the ER. Furthermore, we found that the function of SDF2-ERdj3B-BiP is implicated with the distinct roles of ERECTAf receptors. Our findings establish that the ERECTAf receptor-mediated signaling in stomatal development is ensured by the activities of the ER quality control system, which preferentially maintains the protein abundance of ERECTA and proper subcellular dynamics of ERL1/ERL2, prior to the receptors reaching their destination - the plasma membrane - to execute their functions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Superfície Celular/genética
4.
Mol Cell ; 67(6): 962-973.e5, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28918898

RESUMO

In the endoplasmic reticulum (ER), Ero1 catalyzes disulfide bond formation and promotes glutathione (GSH) oxidation to GSSG. Since GSSG cannot be reduced in the ER, maintenance of the ER glutathione redox state and levels likely depends on ER glutathione import and GSSG export. We used quantitative GSH and GSSG biosensors to monitor glutathione import into the ER of yeast cells. We found that glutathione enters the ER by facilitated diffusion through the Sec61 protein-conducting channel, while oxidized Bip (Kar2) inhibits transport. Increased ER glutathione import triggers H2O2-dependent Bip oxidation through Ero1 reductive activation, which inhibits glutathione import in a negative regulatory loop. During ER stress, transport is activated by UPR-dependent Ero1 induction, and cytosolic glutathione levels increase. Thus, the ER redox poise is tuned by reciprocal control of glutathione import and Ero1 activation. The ER protein-conducting channel is permeable to small molecules, provided the driving force of a concentration gradient.


Assuntos
Retículo Endoplasmático/enzimologia , Proteínas Fúngicas/metabolismo , Glutationa/metabolismo , Glicoproteínas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Canais de Translocação SEC/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/enzimologia , Difusão Facilitada , Proteínas Fúngicas/genética , Dissulfeto de Glutationa/metabolismo , Glicoproteínas/genética , Proteínas de Choque Térmico HSP70/genética , Peróxido de Hidrogênio/metabolismo , Membranas Intracelulares/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Resposta a Proteínas não Dobradas
5.
Cell Mol Life Sci ; 81(1): 299, 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39001944

RESUMO

BACKGROUND: Acetaminophen (APAP) overdose is a significant contributor to drug-induced liver injury worldwide. G-protein-coupled receptor 116 (GPR116) is an important homeostatic maintenance molecule in the body, but little is known about its role in APAP-induced liver injury (AILI). METHODS: GPR116 expression was determined in both human and mouse AILI models. Hepatic function and damage response were analyzed in hepatocyte-specific GPR116 deletion (GPR116△HC) mice undergoing APAP challenge. RNA-sequencing, immunofluorescence confocal, and co-immunoprecipitation (CO-IP) were employed to elucidate the impact and underlying mechanisms of GPR116 in AILI. RESULTS: Intrahepatic GPR116 was upregulated in human and mice with AILI. GPR116△HC mice were vulnerable to AILI compared to wild-type mice. Overexpression of GPR116 effectively mitigated AILI in wild-type mice and counteracted the heightened susceptibility of GPR116△HC mice to APAP. Mechanistically, GPR116 inhibits the binding immunoglobulin protein (BiP), a critical regulator of ER function, through its interaction with ß-arrestin1, thereby mitigating ER stress during the early stage of AILI. Additionally, the activation of GPR116 by ligand FNDC4 has been shown to confer a protective effect against early hepatotoxicity caused by APAP in murine model. CONCLUSIONS: Upregulation of GPR116 on hepatocytes inhibits ER stress by binding to ß-arrestin1, protecting mice from APAP-induced hepatotoxicity. GPR116 may serve as a promising therapeutic target for AILI.


Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Estresse do Retículo Endoplasmático , Receptores Acoplados a Proteínas G , Animais , Humanos , Masculino , Camundongos , Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética
6.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35078937

RESUMO

Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.


Assuntos
Chaperona BiP do Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Camundongos , Dobramento de Proteína
7.
J Biol Chem ; 299(11): 105274, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37739037

RESUMO

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.


Assuntos
Chaperona BiP do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Chaperonas Moleculares , Chaperona BiP do Retículo Endoplasmático/química , Chaperona BiP do Retículo Endoplasmático/genética , Chaperona BiP do Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Células HEK293 , Cadeias J de Imunoglobulina/metabolismo , Domínios Proteicos
8.
Chembiochem ; 25(12): e202300789, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38613462

RESUMO

The human glucose-regulated protein GRP78 is a human chaperone that translocactes to the cell surface when cells are under stress. Theoretical studies suggested it could be involved in SARS-CoV-2 virus entry to cells. In this work, we used in vitro surface plasmon resonance-based assays to show that human GRP78 indeed binds to SARS-CoV-2 spike protein. We have designed and synthesised cyclic peptides based on the loop structure of amino acids 480-488 of the SARS-CoV-2 spike protein S1 domain from the Wuhan and Omicron variants and showed that both peptides bind to GRP78. Consistent with the greater infectiousness of the Omicron variant, the Omicron-derived peptide displays slower dissociation from the target protein. Both peptides significantly inhibit the binding of wild-type S1 protein to the human protein GRP78 suggesting that further development of these cyclic peptide motifs may provide a viable route to novel anti-SARS-CoV-2 agents.


Assuntos
Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Peptídeos Cíclicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/metabolismo , Ligação Proteica , COVID-19/virologia , COVID-19/metabolismo
9.
Pharmacol Res ; 201: 107105, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38367917

RESUMO

Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid ß-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.


Assuntos
Acil-CoA Oxidase , Transplante de Rim , Rim , Doenças Metabólicas , Animais , Ratos , Acil-CoA Oxidase/metabolismo , Aloenxertos , Fibrose , Rim/patologia , Lipídeos
10.
Acta Pharmacol Sin ; 45(2): 378-390, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37798352

RESUMO

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 µM). PE5 (50, 100, 200 µM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.


Assuntos
Neoplasias da Mama , Proteínas de Transporte , Humanos , Animais , Camundongos , Feminino , Proteínas de Transporte/metabolismo , Neoplasias da Mama/tratamento farmacológico , Estresse do Retículo Endoplasmático , Apoptose , Resposta a Proteínas não Dobradas , Proteínas Reguladoras de Apoptose/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo
11.
Int J Mol Sci ; 25(2)2024 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-38279270

RESUMO

The BiP co-chaperone DNAJC3 protects cells during ER stress. In mice, the deficiency of DNAJC3 leads to beta-cell apoptosis and the gradual onset of hyperglycemia. In humans, biallelic DNAJC3 variants cause a multisystem disease, including early-onset diabetes mellitus. Recently, hyperinsulinemic hypoglycemia (HH) has been recognized as part of this syndrome. This report presents a case study of an individual with HH caused by DNAJC3 variants and provides an overview of the metabolic phenotype of individuals with HH and DNAJC3 variants. The study demonstrates that HH may be a primary symptom of DNAJC3 deficiency and can persist until adolescence. Additionally, glycemia and insulin release were analyzed in young DNACJ3 knockout (K.O.) mice, which are equivalent to human infants. In the youngest experimentally accessible age group of 4-week-old mice, the in vivo glycemic phenotype was already dominated by a reduced total insulin secretion capacity. However, on a cellular level, the degree of insulin release of DNAJC3 K.O. islets was higher during periods of increased synthetic activity (high-glucose stimulation). We propose that calcium leakage from the ER into the cytosol, due to disrupted DNAJC3-controlled gating of the Sec61 channel, is the most likely mechanism for HH. This is the first genetic mechanism explaining HH solely by the disruption of intracellular calcium homeostasis. Clinicians should screen for HH in DNAJC3 deficiency and consider DNAJC3 variants in the differential diagnosis of congenital hyperinsulinism.


Assuntos
Hiperinsulinismo Congênito , Proteínas de Choque Térmico HSP40 , Adolescente , Animais , Humanos , Camundongos , Cálcio/metabolismo , Hiperinsulinismo Congênito/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Insulina/metabolismo , Secreção de Insulina , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo
12.
J Biol Chem ; 298(7): 102061, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35609712

RESUMO

The concentration of Ca2+ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca2+ concentrations and the dynamic Ca2+ flux between the different subcellular compartments are tightly controlled. Influx of Ca2+ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase pump, building the Ca2+ gradient across the ER membrane required for ATP import. Meanwhile, Ca2+ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review, we provide an overview of the complex regulation of Ca2+ homeostasis, Ca2+ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca2+ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident cochaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca2+ concentrations.


Assuntos
Sinalização do Cálcio , Cálcio , Citosol , Retículo Endoplasmático , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Homeostase , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo
13.
J Biol Chem ; 298(4): 101537, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35041824

RESUMO

In eukaryotic cells, uptake of cytosolic ATP into the endoplasmic reticulum (ER) lumen is critical for the proper functioning of chaperone proteins. The human transport protein SLC35B1 was recently postulated to mediate ATP/ADP exchange in the ER; however, the underlying molecular mechanisms mediating ATP uptake are not completely understood. Here, we extensively characterized the transport kinetics of human SLC35B1 expressed in yeast that was purified and reconstituted into liposomes. Using [α32P]ATP uptake assays, we tested the nucleotide concentration dependence of ATP/ADP exchange activity on both sides of the membrane. We found that the apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side. Because SLC35B1-containing liposomes were preferentially inside-out oriented, these results suggest a low-affinity external site and a high-affinity internal site in the ER. Three different experimental approaches indicated that ATP/ADP exchange by SLC35B1 was not strict, and that other di- and tri-nucleotides could act as suitable counter-substrates for ATP, although mononucleotides and nucleotide sugars were not transported. Finally, bioinformatic analysis and site-directed mutagenesis identified that conserved residues K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play critical roles in transport. The fact that SLC35B1 can promote ATP transport in exchange for ADP or UDP suggest a more direct coupling between ATP import requirements and the need for eliminating ADP and UDP, which are generated as side products of reactions taking place in the ER-lumen.


Assuntos
Trifosfato de Adenosina , Retículo Endoplasmático , Proteínas de Transporte de Monossacarídeos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Lipossomos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Saccharomyces cerevisiae/genética , Difosfato de Uridina/metabolismo
14.
Lab Invest ; 103(7): 100130, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36925047

RESUMO

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Chaperona BiP do Retículo Endoplasmático , Neoplasias Hepáticas/metabolismo , Estresse do Retículo Endoplasmático , Apoptose , RNA , Proteínas Quinases , Colectinas
15.
EMBO J ; 38(21): e102177, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31531998

RESUMO

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Humanos , Conformação Proteica
16.
J Gen Virol ; 104(4)2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37097881

RESUMO

Swine are considered to be an important intermediate host in the cycle of Japanese encephalitis virus (JEV) infection. Most existing antiviral studies of JEV mainly focus on the host factor of the dead-end hosts. However, little research has addressed this in swine. Here, we found that swine interferon alpha-inducible protein 6 (sIFI6) possessed antiviral activity against JEV. In vitro studies showed that overexpression of sIFI6 inhibited the infection of JEV, while sIFI6 knockdown enhanced the infection of JEV in PK-15 cells. In addition, we also found that the structural integrity of sIFI6 was required by anti-JEV activity and that sIFI6 interacted with JEV nonstructural protein 4A (NS4A), an integral membrane protein with a pivotal function in replication complex during JEV replication. The interaction domain was mapped to the fourth transmembrane domain (TMD), also known as the 2K peptide of NS4A. The antiviral activity of sIFI6 was regulated by endoplasmic reticulum (ER) stress-related protein, Bip. In vivo studies revealed that sIFI6 alleviated symptoms of JEV infection in C57BL/6 mice. In addition, the antiviral spectrum of sIFI6 showed that sIFI6 specifically inhibited JEV infection. In conclusion, this study identified sIFI6 as a host factor against JEV infection for the first time. Our findings provide a potential drug target against JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie) , Encefalite Japonesa , Animais , Camundongos , Antivirais/uso terapêutico , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie)/metabolismo , Encefalite Japonesa/metabolismo , Camundongos Endogâmicos C57BL , Suínos , Replicação Viral , Fosfoproteínas/metabolismo , Proteínas Nucleares/metabolismo
17.
Mol Med ; 29(1): 112, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37605113

RESUMO

Glucose-Regulated Protein 78 (GRP78) is a chaperone protein that is predominantly expressed in the lumen of the endoplasmic reticulum. GRP78 plays a crucial role in protein folding by assisting in the assembly of misfolded proteins. Under cellular stress conditions, GRP78 can translocate to the cell surface (csGRP78) were it interacts with different ligands to initiate various intracellular pathways. The expression of csGRP78 has been associated with tumor initiation and progression of multiple cancer types. This review provides a comprehensive analysis of the existing evidence on the roles of GRP78 in various types of cancer and other human pathology. Additionally, the review discusses the current understanding of the mechanisms underlying GRP78's involvement in tumorigenesis and cancer advancement. Furthermore, we highlight recent innovative approaches employed in downregulating GRP78 expression in cancers as a potential therapeutic target.


Assuntos
Chaperona BiP do Retículo Endoplasmático , Neoplasias , Humanos , Neoplasias/genética , Transformação Celular Neoplásica , Retículo Endoplasmático
18.
Environ Sci Technol ; 57(48): 20169-20181, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-37933956

RESUMO

Submerged munitions from World War I and II are threatening human activities in the oceans, including fisheries and shipping or the construction of pipelines and offshore facilities. To avoid unforeseen explosions, remotely controlled "blast-in-place" (BiP) operations are a common practice worldwide. However, after underwater BiP detonations, the toxic and carcinogenic energetic compounds (ECs) will not completely combust but rather distribute within the marine ecosphere. To shed light on this question, two comparable World War II mines in Denmark's Sejerø Bay (Baltic Sea) were blown up by either low-order or high-order BiP operations by the Royal Danish Navy. Water and sediment samples were taken before and immediately after the respective BiP operation and analyzed for the presence of ECs with sensitive GC-MS/MS and LC-MS/MS technology. EC concentrations increased after high-order BiP detonations up to 353 ng/L and 175 µg/kg in water and sediment, respectively, while low-order BiP detonations resulted in EC water and sediment concentrations up to 1,000,000 ng/L (1 mg/L) and >10,000,000 µg/kg (>10 g/kg), respectively. Our studies provide unequivocal evidence that BiP operations in general lead to a significant increase of contamination of the marine environment and ecotoxicological risk with toxic ECs. Moreover, as compared to high-order BiP detonations, low-order BiP detonations resulted in a several 1000-fold higher burden on the marine environment.


Assuntos
Explosões , Poluentes Químicos da Água , Humanos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Oceanos e Mares , Água , Poluentes Químicos da Água/toxicidade
19.
Cell Mol Life Sci ; 79(8): 414, 2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35816252

RESUMO

Helicobacter pylori-mediated gastric carcinogenesis involves upregulation of the E3 ubiquitin ligase Siah2 and its phosphorylation-mediated stabilization. This study elucidates a novel mechanism of oxidative stress regulation by phosphorylated Siah2 in H. pylori-infected gastric epithelial cancer cells (GECs). We identify that H. pylori-mediated Siah2 phosphorylation at the 6th serine residue (P-S6-Siah2) enhances proteasomal degradation of the 78-kDa glucose-regulated protein (GRP78) possessing antioxidant functions. S6 phosphorylation stabilizes Siah2 and P-S6-Siah2 potentiates H. pylori-mediated reactive oxygen species (ROS) generation. However, infected S6A phospho-null Siah2-expressing cells have decreased cellular GRP78 level as surprisingly these cells release GRP78 to a higher extent and accumulate significantly higher ROS than the wild type (WT) Siah2 construct-expressing cells. Ectopic expression of GRP78 prevents the loss of mitochondrial membrane potential and cellular ROS accumulation caused by H. pylori. H. pylori-induced mitochondrial damage and mitochondrial membrane potential loss are potentiated in Siah2-overexpressing cells but these effects are further enhanced in S6A-expressing cells. This study also confirms that while phosphorylation-mediated Siah2 stabilization optimally upregulates aggresome accumulation, it suppresses autophagosome formation, thus decreasing the dependency on the latter mechanism in regulating cellular protein abundance. Disruption of the phospho-Siah2-mediated aggresome formation impairs proliferation of infected GECs. Thus, Siah2 phosphorylation has diagnostic and therapeutic significance in H. pylori-mediated gastric cancer (GC).


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/fisiologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
20.
Int J Mol Sci ; 24(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36614266

RESUMO

Modern pharmacotherapy of neurodegenerative diseases is predominantly symptomatic and does not allow vicious circles causing disease development to break. Protein misfolding is considered the most important pathogenetic factor of neurodegenerative diseases. Physiological mechanisms related to the function of chaperones, which contribute to the restoration of native conformation of functionally important proteins, evolved evolutionarily. These mechanisms can be considered promising for pharmacological regulation. Therefore, the aim of this review was to analyze the mechanisms of endoplasmic reticulum stress (ER stress) and unfolded protein response (UPR) in the pathogenesis of neurodegenerative diseases. Data on BiP and Sigma1R chaperones in clinical and experimental studies of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease are presented. The possibility of neuroprotective effect dependent on Sigma1R ligand activation in these diseases is also demonstrated. The interaction between Sigma1R and BiP-associated signaling in the neuroprotection is discussed. The performed analysis suggests the feasibility of pharmacological regulation of chaperone function, possibility of ligand activation of Sigma1R in order to achieve a neuroprotective effect, and the need for further studies of the conjugation of cellular mechanisms controlled by Sigma1R and BiP chaperones.


Assuntos
Doenças Neurodegenerativas , Fármacos Neuroprotetores , Humanos , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ligantes , Chaperonas Moleculares/metabolismo , Resposta a Proteínas não Dobradas , Estresse do Retículo Endoplasmático , Doenças Neurodegenerativas/tratamento farmacológico
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