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1.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
2.
Gene ; 803: 145893, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34384864

RESUMO

Glycolysis and heat shock proteins (HSPs) play an important role in mediating the physiological response to hypoxia. The changes of glycolysis and HSPs with altitude would provide important information regarding ways to prevent hypoxia-related sickness in both animals and humans. In this study, the expression pattern of HIF1A, PDK4, HSP27 and HSP60, indexes activity and content of glucose metabolism were detected in heart, lung, brain, and quadriceps femoris taken from Tibetan sheep (Ovis aries) that were raised at different altitudes (2,500 m, 3,500 m and 4,500 m). The expression of HIF1A and PDK4 was increased with increasing altitude in all of the tissues. The lactate dehydrogenase (LDH) activities and adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH (redox state), NAD+), lactic acid (LA), pyruvic acid (PA) contents were all increased with increasing altitude in all of the tissues. The ratio of NADH/NAD+ and LA/PA were higher in sheep at an altitude of 4,500 m than of 3,500 m and 2,500 m in all tissues, except for the NADH/NAD+ ratio in lung and quadriceps femoris. An increase in the protein and mRNA expression of ATP-independent HSP27 during hypoxia condition was detected. The expression of ATP-dependent HSP60 mRNA and protein was increased in all of the tissues at an altitude of 3,500 m than of 2,500 m, but was decreased at an altitude of 4,500 m. These results suggest that glycolysis and HSPs are upregulated to ensure energy supply and proteostasis during hypoxia, but energy conservation may be prioritized over cytoprotective protein chaperoning in Tibetan sheep tissues during extreme hypoxia.


Assuntos
Aclimatação , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Altitude , Animais , Hipóxia Celular , Regulação da Expressão Gênica , Glicólise , Ovinos , Tibet , Regulação para Cima
3.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445306

RESUMO

Epilepsy is a pathologic condition with high prevalence and devastating consequences for the patient and its entourage. Means for accurate diagnosis of type, patient monitoring for predicting seizures and follow up, and efficacious treatment are desperately needed. To improve this adverse outcome, miRNAs and the chaperone system (CS) are promising targets to understand pathogenic mechanisms and for developing theranostics applications. miRNAs implicated in conditions known or suspected to favor seizures such as neuroinflammation, to promote epileptic tolerance and neuronal survival, to regulate seizures, and others showing variations in expression levels related to seizures are promising candidates as useful biomarkers for diagnosis and patient monitoring, and as targets for developing novel therapies. Components of the CS are also promising as biomarkers and as therapeutic targets, since they participate in epileptogenic pathways and in cytoprotective mechanisms in various epileptogenic brain areas, even if what they do and how is not yet clear. The data in this review should help in the identification of molecular targets among the discussed miRNAs and CS components for research aiming at understanding epileptogenic mechanisms and, subsequently, develop means for predicting/preventing seizures and treating the disease.


Assuntos
Epilepsia/metabolismo , Proteínas de Choque Térmico/metabolismo , MicroRNAs/metabolismo , Animais , Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Epilepsia/genética , Epilepsia/patologia , Proteínas de Choque Térmico/genética , Humanos , MicroRNAs/genética
4.
Commun Biol ; 4(1): 829, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211114

RESUMO

To further the understanding of the evolution of transcriptional regulation, we profiled genome-wide transcriptional start sites (TSSs) in two sub-species, Bos taurus taurus and Bos taurus indicus, that diverged approximately 500,000 years ago. Evolutionary and developmental-stage differences in TSSs were detected across the sub-species, including translocation of dominant TSS and changes in TSS distribution. The 16% of all SNPs located in significant differentially used TSS clusters across sub-species had significant shifts in allele frequency (472 SNPs), indicating they may have been subject to selection. In spleen and muscle, a higher relative TSS expression was observed in Bos indicus than Bos taurus for all heat shock protein genes, which may be responsible for the tropical adaptation of Bos indicus.


Assuntos
Bovinos/genética , Variação Genética/genética , Polimorfismo de Nucleotídeo Único , Sítio de Iniciação de Transcrição , Transcrição Genética/genética , Animais , Bovinos/classificação , Evolução Molecular , Expressão Gênica , Frequência do Gene , Especiação Genética , Proteínas de Choque Térmico/genética , Fígado/metabolismo , Músculos/metabolismo , Especificidade de Órgãos/genética , Especificidade da Espécie , Baço/metabolismo
5.
Poult Sci ; 100(8): 101260, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34198090

RESUMO

The present study compared the histochemical and meat quality characteristics of broiler pectoralis major (PM) muscle among the groups categorized according to muscle abnormalities, including pale, soft, and exudative (PSE)-like condition and white-striping (WS) feature. Additionally, this study investigated the associations between muscular abnormalities and expression levels of heat shock proteins (HSPs), including αß-crystallin, HSP70, and HSP90, at the early postmortem period. The WS breasts with normal quality condition showed greater PM muscle weight and were more associated with fiber hypertrophy, compared to the no WS breasts with PSE-like condition (P < 0.05). The PSE-like group exhibited paler surface color and tougher meat, causing more fluid loss after cooking, compared to the normal quality group (P < 0.05). However, there were no significant differences in the quality traits between the WS groups (P > 0.05), except for lightness and cooking loss. Higher αß-crystallin and HSP90 expression levels were observed in PSE-like breast compared to normal quality breast (P < 0.05), whereas WS pattern was not related with HSPs levels (P > 0.05). Therefore, HSP levels at the early postmortem period, especially those of αß-crystallin and HSP90, were associated with the breast quality characteristics of PSE-like condition broilers.


Assuntos
Galinhas , Músculos Peitorais , Animais , Culinária , Proteínas de Choque Térmico/genética , Carne/análise
6.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152408

RESUMO

The development of ovarian follicles constitutes the foundation of female reproduction. The proliferation of granulosa cells (GCs) is a basic process required to ensure normal follicular development. However, the mechanisms involved in controlling GC cell cycle are not fully understood. Here, by performing gene expression profiling in the domestic pig (Sus scrofa), we showed that cell cycle arrest at G0/G1 phase is highly correlated with pathways associated with hypoxic stress and FOXO signalling. Specifically, the elevated proportion of GCs at the arrested G0/G1 phase was accompanied by increased nuclear translocation of FOXO1 under conditions of hypoxia both in vivo and in vitro. Furthermore, phosphorylation of 14-3-3 by the JNK kinase is required for hypoxia-mediated FOXO1 activation and the resultant G0/G1 arrest. Notably, a FOXO1 mutant without DNA-binding activity failed to induce G0/G1 arrest of GCs during hypoxia. Importantly, we identified a new target gene of FOXO1, namely TP53INP1, which contributes to suppression of the G1-S cell cycle transition in response to hypoxia. Furthermore, we demonstrated that the inhibitory effect of the FOXO1-TP53INP1 axis on the GC cell cycle is mediated through a p53-CDKN1A-dependent mechanism. These findings could provide avenues for the clinical treatment of human infertility caused by impaired follicular development.


Assuntos
Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Forkhead Box O1/metabolismo , Proteínas de Choque Térmico/metabolismo , Hipóxia/metabolismo , Folículo Ovariano/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Transporte/genética , Ciclo Celular , Divisão Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Proteína Forkhead Box O1/genética , Fase G1 , Células da Granulosa/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Hipóxia/genética , Fosforilação , Fase de Repouso do Ciclo Celular , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
7.
Nat Commun ; 12(1): 3285, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078899

RESUMO

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.


Assuntos
Nervo Femoral/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Tibial/metabolismo , Animais , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Axônios/metabolismo , Axônios/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Nervo Femoral/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Células de Schwann/patologia , Nervo Isquiático/patologia , Estresse Fisiológico , Nervo Tibial/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
8.
Mar Pollut Bull ; 169: 112545, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34111604

RESUMO

Constant evolution of omics-technologies has provided access to identification of various important gene families. Recently, genome assemblies on widely used ecotoxicological model species, including rotifers and copepods have been completed and representative detoxification-related gene families have been discovered for biomarker genes. However, despite ubiquitous presence of stress-response proteins, limited information on full genome-wide report on heat shock proteins (Hsps) is available. Various studies have demonstrated multiple cellular functions of Hsps in living organisms as an important biomarker in response to abiotic and biotic stressors, however, full genome-wide identification of Hsps, particularly in aquatic invertebrates, has not been reported. This is the first study to report the entire Hsps and basal gene expression levels in three regional-specific copepods: Tigriopus japonicus and kingsejongensis, Paracyclopina nana, and Eurytemora affnis, and how each Hsp family gene is regulated at a basal level.


Assuntos
Copépodes , Proteínas de Choque Térmico , Rotíferos , Animais , Copépodes/genética , Ecotoxicologia , Proteínas de Choque Térmico/genética
9.
Nat Commun ; 12(1): 3480, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108473

RESUMO

Acclimation to high temperature increases plants' tolerance of subsequent lethal high temperatures. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6C loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate these HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.


Assuntos
Arabidopsis/fisiologia , Proteínas de Choque Térmico/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Termotolerância/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Desmetilação , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Mutação
10.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074030

RESUMO

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Hibridização In Situ , Lantânio/farmacologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
11.
Cell Mol Life Sci ; 78(12): 5179-5195, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33974094

RESUMO

Despite new advances on the functions of ER chaperones at the cell surface, the translocation mechanisms whereby these chaperones can escape from the ER to the cell surface are just emerging. Previously we reported that in many cancer types, upon ER stress, IRE1α binds to and triggers SRC activation resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. In this study, using a combination of molecular, biochemical, and imaging approaches, we discovered that in colon and lung cancer, upon ER stress, ER chaperones, such as GRP78 bypass the Golgi and unconventionally traffic to the cell surface via endosomal transport mediated by Rab GTPases (Rab4, 11 and 15). Such unconventional transport is driven by membrane fusion between ER-derived vesicles and endosomes requiring the v-SNARE BET1 and t-SNARE Syntaxin 13. Furthermore, GRP78 loading into ER-derived vesicles requires the co-chaperone DNAJC3 that is regulated by ER-stress induced PERK-AKT-mTOR signaling.


Assuntos
Membrana Celular/metabolismo , Neoplasias do Colo/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutagênese Sítio-Dirigida , Mutação , Transporte Proteico , Transdução de Sinais , Células Tumorais Cultivadas
12.
J Bacteriol ; 203(14): e0014321, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33941609

RESUMO

Proteolysis is a fundamental property of all living cells. In the bacterium Salmonella enterica serovar Typhimurium, the HspQ protein controls the specificities of the Lon and ClpAP proteases. Upon acetylation, HspQ stops being a Lon substrate and no longer enhances proteolysis of the Lon substrate Hha. The accumulated HspQ protein binds to the protease adaptor ClpS, hindering proteolysis of ClpS-dependent substrates of ClpAP, such as Oat, a promoter of antibiotic persistence. HspQ is acetylated by the protein acetyltransferase Pat from acetyl coenzyme A (acetyl-CoA) bound to the acetyl-CoA binding protein Qad. We now report that low cytoplasmic Mg2+ promotes qad expression, which protects substrates of Lon and ClpSAP by increasing HspQ amounts. The qad promoter is activated by PhoP, a regulatory protein highly activated in low cytoplasmic Mg2+ that also represses clpS transcription. Both the qad gene and PhoP repression of the clpS promoter are necessary for antibiotic persistence. PhoP also promotes qad transcription in Escherichia coli, which shares a similar PhoP box in the qad promoter region with S. Typhimurium, Salmonella bongori, and Enterobacter cloacae. Our findings identify cytoplasmic Mg2+ and the PhoP protein as critical regulators of protease specificity in multiple enteric bacteria. IMPORTANCE The bacterium Salmonella enterica serovar Typhimurium narrows down the spectrum of substrates degraded by the proteases Lon and ClpAP in response to low cytoplasmic Mg2+, a condition that decreases protein synthesis. This control is exerted by PhoP, a transcriptional regulator activated in low cytoplasmic Mg2+ that governs proteostasis and is conserved in enteric bacteria. The uncovered mechanism enables bacteria to control the abundance of preexisting proteins.


Assuntos
Citoplasma/metabolismo , Magnésio/metabolismo , Protease La/metabolismo , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citoplasma/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Protease La/química , Protease La/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por Substrato
13.
J Biol Chem ; 296: 100759, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33965375

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Proteínas de Choque Térmico/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Resposta a Proteínas não Dobradas , Células Vero
14.
Arch Virol ; 166(8): 2089-2108, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33934196

RESUMO

The SARS-CoV-2 pandemic has become one of the most serious health concerns globally. Although multiple vaccines have recently been approved for the prevention of coronavirus disease 2019 (COVID-19), an effective treatment is still lacking. Our knowledge of the pathogenicity of this virus is still incomplete. Studies have revealed that viral factors such as the viral load, duration of exposure to the virus, and viral mutations are important variables in COVID-19 outcome. Furthermore, host factors, including age, health condition, co-morbidities, and genetic background, might also be involved in clinical manifestations and infection outcome. This review focuses on the importance of variations in the host genetic background and pathogenesis of SARS-CoV-2. We will discuss the significance of polymorphisms in the ACE-2, TMPRSS2, vitamin D receptor, vitamin D binding protein, CD147, glucose-regulated protein 78 kDa, dipeptidyl peptidase-4 (DPP4), neuropilin-1, heme oxygenase, apolipoprotein L1, vitamin K epoxide reductase complex 1 (VKORC1), and immune system genes for the clinical outcome of COVID-19.


Assuntos
COVID-19/genética , Sistema ABO de Grupos Sanguíneos/genética , Enzima de Conversão de Angiotensina 2/genética , Apolipoproteína L1/genética , Basigina/genética , COVID-19/epidemiologia , COVID-19/terapia , Dipeptidil Peptidase 4/genética , Proteínas de Choque Térmico/genética , Heme Oxigenase-1/genética , Humanos , Imunidade/genética , Neuropilina-1/genética , Avaliação de Resultados da Assistência ao Paciente , Polimorfismo Genético , Receptores de Calcitriol/genética , SARS-CoV-2 , Serina Endopeptidases/genética , Proteína de Ligação a Vitamina D/genética , Vitamina K Epóxido Redutases/genética
15.
Biochemistry ; 60(17): 1356-1367, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33881310

RESUMO

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a non-ideal coiled coil. Partial unfolding of this region during heat stress results in reduced nucleotide exchange and disrupts protein folding by DnaK. In this study, we elucidate the role of non-ideality in the coiled-coil domain of Escherichia coli GrpE in controlling its co-chaperone activity. The presence of a four-residue stutter introduces nonheptad periodicity in the GrpE coiled coil, resulting in global structural changes in GrpE and regulating its interaction with DnaK. Introduction of hydrophobic residues at the stutter core increased the structural stability of the protein. Using an in vitro FRET assay, we show that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, these mutants were unable to support bacterial growth at 42°C in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in GrpE in regulating the DnaK-chaperone cycle during heat stress. More generally, our findings illustrate how stutters in a coiled-coil domain regulate structure-function trade-off in proteins.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Modelos Moleculares , Mutação , Domínios Proteicos
16.
PLoS Genet ; 17(4): e1009521, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33857138

RESUMO

The RNA-binding protein Mrn1 in Saccharomyces cerevisiae targets over 300 messenger RNAs, including many involved in cell wall biogenesis. The impact of Mrn1 on these target transcripts is not known, however, nor is the cellular role for this regulation. We have shown that Mrn1 represses target mRNAs through the action of its disordered, asparagine-rich amino-terminus. Its endogenous targets include the paralogous SUN domain proteins Nca3 and Uth1, which affect mitochondrial and cell wall structure and function. While loss of MRN1 has no effect on fermentative growth, we found that mrn1Δ yeast adapt more quickly to respiratory conditions. These cells also have enlarged mitochondria in fermentative conditions, mediated in part by dysregulation of NCA3, and this may explain their faster switch to respiration. Our analyses indicated that Mrn1 acts as a hub for integrating cell wall integrity and mitochondrial biosynthesis in a carbon-source responsive manner.


Assuntos
Parede Celular/genética , Proteínas de Choque Térmico/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Parede Celular/metabolismo , Homeostase/genética , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/biossíntese , Biogênese de Organelas , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
17.
PLoS Genet ; 17(4): e1009539, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33914734

RESUMO

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.


Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/genética , Substituição de Aminoácidos/genética , Doença de Canavan/patologia , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
18.
J Biol Chem ; 296: 100660, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862083

RESUMO

The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasome-activating nucleotidase proline residue (P91) forms cis- and trans-peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5-P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2-P103A rpt5-P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperone-mediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.


Assuntos
Proteínas de Choque Térmico/metabolismo , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteínas de Choque Térmico/genética , Mutação , Prolina/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Domínios Proteicos , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Ubiquitina-Proteína Ligases/genética
19.
Aging (Albany NY) ; 13(8): 11774-11785, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33883304

RESUMO

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, accounting for 50-70% of anovulatory infertility cases. However, the etiology of PCOS at the molecular level remains unclear. Here, bioinformatics analysis was performed to identify differentially expressed genes (DEGs) between adipose tissue of PCOS patients and matched tissues from non-hyperandrogenic women. RT-qPCR, western blot, cell counting kit-8 (CCK-8), EdU (5-Ethynyl-2'-deoxyuridine) staining, LC3 staining, ROS (reactive oxygen species) detection, and apoptosis assays were conducted to explore the effects of sestrin 1 on KGN human granulosa-like tumor cells. Bioinformatics analysis indicated that DEGs in adipose tissue from PCOS patients were enriched in the p53 signaling pathway. Moreover, sestrin 1 was identified as a major target of the p53 gene. Downregulation of sestrin 1 inhibited proliferation of KGN cells by inhibiting autophagy. Additionally, sestrin 1 downregulation increased ROS generation and promoted apoptosis in KGN cells. By contrast, overexpression of sestrin 1 increased cell viability by increasing autophagy in KGN cells. Together, these results suggest that downregulation of sestrin 1 may be a potential novel treatment strategy for PCOS.


Assuntos
Autofagia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Choque Térmico/genética , Gordura Intra-Abdominal/patologia , Síndrome do Ovário Policístico/genética , Animais , Apoptose/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Biologia Computacional , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células da Granulosa/patologia , Voluntários Saudáveis , Humanos , Omento , Síndrome do Ovário Policístico/patologia , Ratos , Espécies Reativas de Oxigênio , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima
20.
Aging (Albany NY) ; 13(8): 11234-11256, 2021 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-33820869

RESUMO

Cerebral ischemia/reperfusion injury causes a series of intricate cascade reactions in brain tissue causing apoptosis and proinflammatory programmed cell death known as pyroptosis of nerve cells. The dysfunction of target organelle mitochondria plays a key role in the process of neuronal apoptosis and pyroptosis. Mesenchymal stem cells (MSCs) have been widely used in the experimental or clinical treatment of various ischemic diseases, but the therapeutic efficacy of MSCs on cerebral ischemia-reperfusion injury need to be improved. We successfully cultured olfactory mucosa MSCs (OM-MSCs) to obtain a better source of seed cells. In this way, the therapeutic potential of OM-MSCs transplantation has been evaluated for ischemic stroke using an optimized culture scheme in vitro. Ischemic-hypoxic preconditioned OM-MSCs (IhOM-MSCs) were used to treat a neuron model of oxygen-glucose deprivation/reperfusion and the middle cerebral artery occlusion in rats. These results demonstrated that IhOM-MSCs mediated the upregulation of the downstream target genes GRP78 and Bcl-2 by miR-181a to protect mitochondrial function and inhibit apoptosis and pyroptosis of neurons in the ischemia/reperfusion injury model. Thus, IhOM-MSCs transplantation may be an effective therapy of ischemic stroke in the future.


Assuntos
Precondicionamento Isquêmico/métodos , AVC Isquêmico/terapia , Transplante de Células-Tronco Mesenquimais/métodos , MicroRNAs/metabolismo , Traumatismo por Reperfusão/terapia , Animais , Apoptose , Encéfalo/citologia , Encéfalo/patologia , Modelos Animais de Doenças , Proteínas de Choque Térmico/genética , Humanos , AVC Isquêmico/complicações , AVC Isquêmico/patologia , Masculino , Mitocôndrias/patologia , Neurônios/citologia , Neurônios/patologia , Mucosa Olfatória/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Traumatismo por Reperfusão/etiologia
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