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1.
Gene ; 736: 144416, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32006595

RESUMO

BACKGROUND: Psoriasis has a complex genetic background with a strong heritable component. Herein, the present meta-analysis aims to evaluate the association of ERAP1 polymorphisms with psoriasis susceptibility. METHODS: PubMed, Web of Sciences, Scopus, and Cochrane Library databases were examined with no time limits up to March 2019, without language, age, and sex restrictions. The odds ratio (OR) and 95% confidence interval (CI) were calculated by CMA 2.0 software in a dichotomous analysis using computed effect sizes and having OR and confidence limits for each study. The subgroup analysis based on ethnicity, type of study, and genotyping method was performed. RESULTS: Thirteen articles were involved in the meta-analysis, in details eight were cohort studies and five were case-control studies. The results showed an association between rs27524 [OR = 1.179; 95%CI: 1.081, 1.286; p < 0.001] and rs30187 [OR = 1.237; 95%CI: 1.133, 1.351; p < 0.001] polymorphisms and psoriasis susceptibility; whereas no association was detected with rs26653 [OR = 1.013; 95%CI: 0.798, 1.286; p = 0.914] and rs27044 [OR = 1.164; 95%CI: 0.982, 1.381; p = 0.080] polymorphisms. Psoriasis susceptibility in both Caucasian and Asian ethnicities was related to rs27524 polymorphism, while rs30187 and rs27044 polymorphisms were over-represented in patients belonging to Caucasian ethnicity. In addition, in cohort studies, psoriasis susceptibility was related to rs27524 polymorphism, while the associated polymorphisms were rs26653 and rs27044 in case-control studies, and rs30187 in both cohort and case-control studies. CONCLUSIONS: These findings showed an association between rs27524 and rs30187 polymorphisms and susceptibility to psoriasis, while lack of association was obtained for rs26653 and rs27044 polymorphisms. In order to confirm our results, further studies are needed, also considering different factors, such as type of psoriasis and ethnicity.


Assuntos
Aminopeptidases/genética , Retículo Endoplasmático/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Psoríase/genética , Animais , Humanos
2.
Nat Commun ; 11(1): 781, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034123

RESUMO

Recent evidence demonstrates that novel protein-coding genes can arise de novo from non-genic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of non-genic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Here, we systematically characterize how these de novo emerging coding sequences impact fitness in budding yeast. Disruption of emerging sequences is generally inconsequential for fitness in the laboratory and in natural populations. Overexpression of emerging sequences, however, is enriched in adaptive fitness effects compared to overexpression of established genes. We find that adaptive emerging sequences tend to encode putative transmembrane domains, and that thymine-rich intergenic regions harbor a widespread potential to produce transmembrane domains. These findings, together with in-depth examination of the de novo emerging YBR196C-A locus, suggest a novel evolutionary model whereby adaptive transmembrane polypeptides emerge de novo from thymine-rich non-genic regions and subsequently accumulate changes molded by natural selection.


Assuntos
Evolução Molecular , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Timina , Fator de Transcrição TFIID/genética , Adaptação Biológica/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica , Aptidão Genética , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Fases de Leitura Aberta , Domínios Proteicos/genética , Saccharomyces cerevisiae/genética
3.
Life Sci ; 247: 117446, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32081662

RESUMO

AIMS: Previous studies showed a close relationship between obesity and asthma. In this study, we investigated the expression of endoplasmic reticulum (ER) stress genes in the lung tissue of obese ovalbumin (OVA)-sensitized male and female rats. MAIN METHODS: The rats were divided into eight groups (n = 5 per group) as follows: female and male rats fed with normal diet (FND and MND, respectively), female and male OVA-sensitized rats fed with normal diet (F-OND and M-OND, respectively), female and male rats fed with high-fat diet (F-HFD and M-HFD, respectively), female and male OVA-sensitized rats fed with high-fat diet (F-OHFD and M-OHFD, respectively). All rats were fed with a high-fat diet or standard pelts for 8 weeks, and for another 4 weeks, they were sensitized by OVA or saline. At the end of the study, lung tissue NF-kB protein level was assessed, and ER stress markers genes expression was determined by Real Time-PCR. KEY FINDING: OVA-sensitization and diet-induced obesity caused the curve of methacholine concentration-response to shift to the left. In addition, the results indicated that the EC50 (the effective concentration of methacholine generating 50% of peak response) in F-OHFD rats was statistically lower than that of the M-OHFD group (p < 0.05). Moreover, the results showed that diet-induced obesity increased the expression of ATF4, ATF6, GRP78, XBP-1, and CHOP as well as the protein level of NF-kB in this experimental model of asthma, markedly in the F-OHFD group. SIGNIFICANCE: The results suggest that ER stress may be involved in the pathogenesis of asthma observed in obese OVA-sensitized rats, especially in the female animals.


Assuntos
Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Leptina/metabolismo , NF-kappa B/metabolismo , Ovalbumina/metabolismo , Animais , Asma/metabolismo , Dieta Hiperlipídica , Retículo Endoplasmático/genética , Feminino , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Pulmão , Masculino , Proteínas de Membrana/metabolismo , Cloreto de Metacolina/metabolismo , NF-kappa B/genética , Obesidade/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Fatores de Tempo
4.
DNA Cell Biol ; 39(2): 226-234, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31895584

RESUMO

Hunter's syndrome (mucopolysaccharidosis type II) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene. Motivated by the case of a child affected by this syndrome, we compared the intracellular fate of wild-type IDS (IDSWT) and four nonsense mutations of IDS (IDSL482X, IDSY452X, IDSR443X, and IDSW337X) generating progressively shorter forms of IDS associated with mild to severe forms of the disease. Our analyses revealed formylation of all forms of IDS at cysteine 84, which is a prerequisite for enzymatic activity. After formylation, IDSWT was transported within lysosomes, where it was processed in the mature form of the enzyme. The length of disease-causing deletions correlated with gravity of the folding and transport phenotype, which was anticipated by molecular dynamics analyses. The shortest form of IDS, IDSW337X, was retained in the endoplasmic reticulum (ER) and degraded by the ubiquitin-proteasome system. IDSR443X, IDSY452X, and IDSL482X passed ER quality control and were transported to the lysosomes, but failed lysosomal quality control, resulting in their rapid clearance and in loss-of-function phenotype. Failure of ER quality control inspection is an established cause of loss of function observed in protein misfolding diseases. Our data reveal that fulfillment of ER requirements might not be sufficient, highlight lysosomal quality control as the distal station to control lysosomal enzymes fitness and pave the way for alternative therapeutic interventions.


Assuntos
Códon sem Sentido/genética , Retículo Endoplasmático/genética , Iduronato Sulfatase/genética , Lisossomos/metabolismo , Mucopolissacaridose II/genética , Animais , Retículo Endoplasmático/metabolismo , Glicoproteínas/genética , Humanos , Camundongos , Mucopolissacaridose II/tratamento farmacológico , Mutação/genética
5.
J Agric Food Chem ; 67(41): 11428-11435, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31589037

RESUMO

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo
6.
PLoS Biol ; 17(10): e3000512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658248

RESUMO

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.


Assuntos
Arrestinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Nucleosídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tiamina/metabolismo , Arrestinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Endocitose/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Proteínas de Transporte de Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
7.
Nat Commun ; 10(1): 3960, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481663

RESUMO

Translation is a basic cellular process and its capacity is adapted to cell function. In particular, secretory cells achieve high protein synthesis levels without triggering the protein stress response. It is unknown how and when translation capacity is increased during differentiation. Here, we show that the transcription factor Creb3l2 is a scaling factor for translation capacity in pituitary secretory cells and that it directly binds ~75% of regulatory and effector genes for translation. In parallel with this cell-autonomous mechanism, implementation of the physiological UPR pathway prevents triggering the protein stress response. Knockout mice for Tpit, a pituitary differentiation factor, show that Creb3l2 expression and its downstream regulatory network are dependent on Tpit. Further, Creb3l2 acts by direct targeting of translation effector genes in parallel with signaling pathways that otherwise regulate protein synthesis. Expression of Creb3l2 may be a useful means to enhance production of therapeutic proteins.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Hipófise/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipófise/citologia , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genética , Proteína 1 de Ligação a X-Box/metabolismo , Xenopus laevis
8.
Nat Commun ; 10(1): 3645, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409786

RESUMO

Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Here, we reveal Mul1-Mfn2 pathway that maintains neuronal mitochondrial integrity under stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mito tethering antagonist. Reduced ER-Mito coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mito anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mito contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction and altered ER-Mito interplay.


Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cálcio/metabolismo , Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Neurônios/citologia , Ubiquitina-Proteína Ligases/genética
9.
Mol Cell ; 75(5): 1058-1072.e9, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31375263

RESUMO

The endoplasmic reticulum (ER) is susceptible to wear-and-tear and proteotoxic stress, necessitating its turnover. Here, we show that the N-degron pathway mediates ER-phagy. This autophagic degradation initiates when the transmembrane E3 ligase TRIM13 (also known as RFP2) is ubiquitinated via the lysine 63 (K63) linkage. K63-ubiquitinated TRIM13 recruits p62 (also known as sequestosome-1), whose complex undergoes oligomerization. The oligomerization is induced when the ZZ domain of p62 is bound by the N-terminal arginine (Nt-Arg) of arginylated substrates. Upon activation by the Nt-Arg, oligomerized TRIM13-p62 complexes are separated along with the ER compartments and targeted to autophagosomes, leading to lysosomal degradation. When protein aggregates accumulate within the ER lumen, degradation-resistant autophagic cargoes are co-segregated by ER membranes for lysosomal degradation. We developed synthetic ligands to the p62 ZZ domain that enhance ER-phagy for ER protein quality control and alleviate ER stresses. Our results elucidate the biochemical mechanisms and pharmaceutical means that regulate ER homeostasis.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteólise , Proteína Sequestossoma-1/metabolismo , Animais , Proteínas de Transporte/genética , Retículo Endoplasmático/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteína Sequestossoma-1/genética , Ubiquitinação
10.
Mol Cell ; 75(5): 996-1006.e8, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31377116

RESUMO

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.


Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae
11.
RNA ; 25(12): 1661-1672, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31455610

RESUMO

Brefeldin A resistance factor 1 (Bfr1p) is a nonessential RNA-binding protein and multicopy suppressor of brefeldin A sensitivity in Saccharomyces cerevisiae Deletion of BFR1 leads to multiple defects, including altered cell shape and size, change in ploidy, induction of P-bodies and chromosomal missegregation. Bfr1p has been shown to associate with polysomes, binds to several hundred mRNAs, and can target some of them to P-bodies. Although this implies a role of Bfr1p in translational control of mRNAs, its molecular function remains elusive. In the present study, we show that mutations in RNA-binding residues of Bfr1p impede its RNA-dependent colocalization with ER, yet do not mimic the known cellular defects seen upon BFR1 deletion. However, a Bfr1 RNA-binding mutant is impaired in binding to ERG4 mRNA, which encodes an enzyme required for the final step of ergosterol biosynthesis. Consistently, bfr1Δ strains show a strong reduction in Erg4p protein levels, most likely because of degradation of misfolded Erg4p. Polysome profiling of bfr1Δ or bfr1 mutant strains reveals a strong shift of ERG4 mRNA to polysomes, consistent with a function of Bfr1p in elongation or increased ribosome loading. Collectively, our data reveal that Bfr1 has at least two separable functions: one in RNA binding and cotranslational protein translocation into the ER and one in ploidy control or chromosome segregation.


Assuntos
Retículo Endoplasmático/genética , Oxirredutases/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Proteínas Repressoras/metabolismo , Leveduras/genética , Leveduras/metabolismo , Transporte Proteico , Transporte de RNA , Proteínas de Ligação a RNA , Ribossomos/metabolismo
12.
PLoS Genet ; 15(7): e1008248, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31260446

RESUMO

The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted proteins. However, studies have shown that mSMP recruitment to the ER in eukaryotes can occur in a manner that is independent of the ribosome, translational control, and the signal recognition particle, although the mechanism remains largely unknown. Here, we identify a cis-acting RNA sequence motif that enhances mSMP localization to the ER and appears to increase mRNA stability, and both the synthesis and secretion of secretome proteins. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif is enriched in mRNAs encoding secretome proteins translated on the ER in eukaryotes and on the inner membrane of prokaryotes. SECReTE consists of ≥10 nucleotide triplet repeats enriched with pyrimidine (C/U) every third base (i.e. NNY, where N = any nucleotide, Y = pyrimidine) and can be present in the untranslated as well as the coding regions of the mRNA. Synonymous mutations that elevate the SECReTE count in a given mRNA (e.g. SUC2, HSP150, and CCW12) lead to an increase in protein secretion in yeast, while a reduction in count led to less secretion and physiological defects. Moreover, the addition of SECReTE to the 3'UTR of an mRNA for an exogenously expressed protein (e.g. GFP) led to its increased secretion from yeast cells. Thus, SECReTE constitutes a novel RNA motif that facilitates ER-localized mRNA translation and protein secretion.


Assuntos
Proteínas Fúngicas/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Retículo Endoplasmático/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Motivos de Nucleotídeos , Biossíntese de Proteínas , Estabilidade de RNA , Transporte de RNA , RNA Fúngico/química , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Mutação Silenciosa
13.
Nat Commun ; 10(1): 2902, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263173

RESUMO

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolite-modulated targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduces the size of a subset of LDs while over-expression of SEIP-1 has the opposite effect. Ultrastructural analysis reveals SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purifies with LDs. Analyses of C. elegans and bacterial genetic mutants indicate a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologously expressed SEIP-1 engages nascent lipid droplets and promotes their subsequent expansion in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by promoting LD diversity.


Assuntos
Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Transporte Proteico
14.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180066

RESUMO

Ischemia-reperfusion (IR) is one of the significant medical problems in China. Triphenyltetrazolium chloride (TTC) staining is used to detect the status of the infarct size, and real-time PCR and western blotting are used to detect expressions of genes. TUNEL assay has been used to detect apoptosis. Using a tree shrew myocardial IR model, we found that in the reperfusion period, resina draconis (RD) treatment reduced the infarct size by TTC staining, and significantly enhanced the superoxide dismutase expression and down-regulated the malondialdehyde concentration in a dose-dependent manner. In hearts showing IR, Bax was increased and Bcl-2 was reduced, and RD treatment inhibited the IR-induced Bax expression and up-regulated the IR suppressed level of Bcl-2. TUNEL assay showed that IR induced the apoptosis of myocardial cells, and RD treatment suppressed the IR-induced apoptosis. CHOP and GRP78 were also upregulated in IR hearts, and RD treatment could significantly attenuate the CHOP and GRP78 levels compared with IR group. We further found that IR decreased the miR-423-3p expression and upregulated its target gene ERK both in mRNA and protein levels, and RD treatment upregulated miR-423-3p expression and downregulated ERK expression compared with the IR group. Importantly, miR-423-3p mimics inhibited IR increased ERK, CHOP and GRP78 expressions, and enhanced IR decreased Bcl-2 expression, and inhibited the IR-induced apoptosis of myocardial cells. The findings of this study suggest that RD treatment inhibited the endoplasmic reticulum induced apoptosis of myocardial cells via regulating miR-423-3p/ERK signaling pathway in a tree shrew myocardial IR model.


Assuntos
Cardiotônicos/farmacologia , Dracaena/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Resinas Vegetais/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Cardiotônicos/isolamento & purificação , Modelos Animais de Doenças , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Malondialdeído/antagonistas & inibidores , Malondialdeído/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Resinas Vegetais/isolamento & purificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tupaiidae , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
15.
Mol Cell ; 75(3): 442-456.e4, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176671

RESUMO

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.


Assuntos
Proteínas de Transporte/genética , Diabetes Mellitus/genética , Proteínas de Choque Térmico HSP70/genética , Insulina/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proinsulina/genética , Autofagia/genética , Diabetes Mellitus/patologia , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Insulina/biossíntese , Mutação/genética , Proinsulina/biossíntese , Agregados Proteicos/genética , Dobramento de Proteína , RNA Interferente Pequeno/genética
16.
Elife ; 82019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31237564

RESUMO

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Diester Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo Ligases/química , Processamento de RNA/genética , Estabilidade de RNA/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Chaetomium/química , Chaetomium/enzimologia , Cristalografia por Raios X , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica , Cinética , Diester Fosfórico Hidrolases/genética , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo Ligases/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas/genética
17.
Nat Cell Biol ; 21(6): 755-767, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110288

RESUMO

Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.


Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Metabolismo Energético , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética
18.
Mol Carcinog ; 58(9): 1623-1630, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31041814

RESUMO

Cancer is associated with a number of conditions such as hypoxia, nutrient deprivation, cellular redox, and pH changes that result in accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) and trigger a stress response known as the unfolded protein response (UPR). The UPR is a conserved cellular survival mechanism mediated by the ER transmembrane proteins activating transcription factor 6, protein kinase-like endoplasmic reticulum kinase, and inositol-requiring enzyme 1α (IRE1α) that act to resolve ER stress and promote cell survival. IRE1α is a kinase/endoribonuclease (RNase) with multiple activities including unconventional splicing of the messenger RNA (mRNA) for the transcription factor X-Box Binding Protein 1 (XBP1), degradation of other mRNAs in a process called regulated IRE1α-dependent decay (RIDD) and activation of a pathway leading to c-Jun N-terminal kinase phosphorylation. Each of these outputs plays a role in the adaptive and cell death responses to ER stress. Many studies indicate an important role for XBP1 and RIDD functions in cancer and new studies suggest that these two functions of the IRE1α RNase can have opposing functions in the early and later stages of cancer pathogenesis. Finally, as more is learned about the context-dependent role of IRE1α in cancer development, specific small molecule inhibitors and activators of IRE1α could play an important role in counteracting the protective shield provided by ER stress signaling in cancer cells.


Assuntos
Endorribonucleases/genética , Regulação da Expressão Gênica/genética , Neoplasias/genética , Resposta a Proteínas não Dobradas/genética , Animais , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Humanos , RNA Mensageiro/genética , Transdução de Sinais/genética
19.
Methods Mol Biol ; 1967: 149-163, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069769

RESUMO

Endoplasmic reticulum protein 5 (ERp5) is a member of the thiol isomerase family of enzymes, whose prototype member is protein disulphide isomerase (PDI). Thiol isomerases catalyze reduction/oxidation (redox) reactions which lead to the cleavage, formation, or isomerization of disulphide bonds in protein substrates. Thiol isomerase reactions on protein disulphides are important for the correct folding of proteins in the endoplasmic reticulum and for the regulation of various protein functions in the extracellular space. Apart from the disulphide reactions, thiol isomerases assist protein folding by chaperone activity.The disulphide redox activity of ERp5 can be measured with functional assays involving artificial or natural substrates containing disulphide bonds. Herein we describe step-by-step assays of ERp5 reductase, isomerization, and de-nitrosylation activity. Disulphide reductase assays include insulin or di-eosin-GSSG as substrates whereas the isomerization assay includes RNase as substrate. The reduction of natural substrates, i.e., integrin αIIbß3, can be detected using maleimide labels of free thiols and Western blotting. The biotin switch assay is used to measure the de-nitrosylation of S-nitrosylated substrates. These assays can measure the activity of purified ERp5 protein but can also be applied for the measurement of thiol isomerase activity in cellular samples.


Assuntos
Dissulfetos/química , Isomerases de Dissulfetos de Proteínas/química , Animais , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Humanos , Ligação Proteica/genética , Dobramento de Proteína , Compostos de Sulfidrila/química
20.
Cell Physiol Biochem ; 52(4): 850-868, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958660

RESUMO

BACKGROUND/AIMS: Endoplasmic reticulum (ER)-resident proteins with a C-terminal KDEL ERretention sequence are captured in the Golgi apparatus by KDEL receptors (KDELRs). The binding of such proteins to these receptors induces their retrograde transport. Nevertheless, some KDEL proteins, such as Protein Disulfide Isomerases (PDIs), are found at the cell surface. PDIs target disulfide bridges in the extracellular domains of proteins, such as integrins or A Disintegrin And Metalloprotease 17 (ADAM17) leading to changes in the structure and function of these molecules. Integrins become activated and ADAM17 inactivated upon disulfide isomerization. The way that PDIs escape from retrograde transport and reach the plasma membrane remains far from clear. Various mechanisms might exist, depending on whether a local cell surface association or a more global secretion is required. METHODS: To get a more detailed insight in the transport of PDIs to the cell surface, methods such as cell surface biotinylation, flow cytometric analysis, immunoprecipitation, fluorescence microscopy as well as labeling of cells with fluorescence labled recombinant PDIA6 was performed. RESULTS: Here, we show that the C-terminal KDEL ER retention sequence is sufficient to prevent secretion of PDIA6 into the extracellular space but is mandatory for its association with the cell surface. The cell surface trafficking of PDIA1, PDIA3, and PDIA6 is dependent on KDELR1, which travels in a dynamic manner to the cell surface. This transport is assumed to result in PDI cell surface association, which differs from PDI inducible secretion into the extracellular space. Distinct PDIs differ in their trafficking properties. Endogenous KDELR1, detectable at the cell surface, might be involved not only in the transport of cell-surface-associated PDIs, but also in their retrieval and internalization from the extracellular space. CONCLUSION: Beside their ER retention motive PDIs travel to the cell surface. Here they target different proteins to render their function. To escape the ER PDIs travel via various pathways. One of them depends on the KDELR1, which can transport its target to the cell surface, where it is to be expected to release its cargo in close vicinity to its target molecules. Hence, the KDEL sequence is needed for cell surface association of PDIs, such as PDIA6.


Assuntos
Proteína ADAM17/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Peptídeos/metabolismo , Proteína ADAM17/genética , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Isomerases de Dissulfetos de Proteínas/genética , Transporte Proteico/fisiologia , Receptores de Peptídeos/genética
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