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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34378783

RESUMO

When the temperature is increased, the heat-shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilised for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes and morphologies in over 2100 electron micrographs, a major restructuring of the internal architecture of the cell during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of the morphology of some organelles (such as the vacuole) were observed. Multivesicular bodies grew by almost 70%, indicating a previously unknown involvement in the heat-shock response. A previously undescribed electron-translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Citoplasma , Resposta ao Choque Térmico , Temperatura Alta , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos
2.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34290138

RESUMO

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope.


Assuntos
Ácido Azetidinocarboxílico/toxicidade , Resposta ao Choque Térmico , Membrana Nuclear/fisiologia , Dobramento de Proteína , Proteostase/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Arsenitos/toxicidade , Peróxido de Hidrogênio/toxicidade , Membrana Nuclear/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Compostos de Sódio/toxicidade , Ubiquitina/metabolismo , Ubiquitinação
3.
J Biol Chem ; 298(11): 102476, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36096201

RESUMO

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing.


Assuntos
Doenças Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Humanos , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Doenças Neurodegenerativas/metabolismo , Dobramento de Proteína , Proteínas de Choque Térmico/metabolismo , Guanilato Quinases/metabolismo
4.
J Pediatr Orthop ; 41(1): e7-e13, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32804861

RESUMO

BACKGROUND: Treatment of symptomatic hip dysplasia in skeletally mature patients with spastic cerebral palsy (CP) can be challenging. This study examines our technical experience with the Bernese periacetabular osteotomy (PAO) in combination with adjunctive procedures in the treatment of this complex hip deformity. METHODS: Sixteen consecutive patients (18 hips) with symptomatic CP hip dysplasia were treated with a PAO and variable adjunctive procedures and retrospectively reviewed. Two patient (2 hips) were excluded due to insufficient follow-up. The average age at the time of surgery was 17.7 years (range: 13 to 28 y). We compared the preoperative to postoperative changes in radiographic parameters as well as early outcomes as measured by patient assessment of hip pain and function using the modified Harris Hip Score (mHHS). RESULTS: The average time of follow-up was 3.3 years (range: 2.0 to 6.3 y). Tönnis angles decreased from a median of 30 degrees (range: 18 to 45 degrees) preoperatively to a median of 6 degrees (range: -9 to 21 degrees) postoperatively. Lateral center-edge angles increased from a median of -8 degrees (range: -28 to 15 degrees) to a median of 32 degrees (range: 19 to 38 degrees). Anterior center-edge angles increased from a median of 2 degrees (range: -22 to 39 degrees) to a median of 35 degrees (range: 22 to 47 degrees). The extrusion index decreased from a median of 57% preoperatively (range: 35% to 73%) to a median of 21% (range: 11% to 36%) postoperatively.The median mHHS was 62 (range: 37 to 81) preoperatively and 85 (range: 65 to 100) postoperatively. Notably, the pain component of the mHHS improved from 20 (range: 0 to 44) to 42 (range: 30 to 44). Tönnis osteoarthritis grade preoperatively was either 0 (11 hips) or 1 (5 hips) and remained unchanged in 11 hips and increased by 1 grade in 5 hips. CONCLUSIONS: It has been our experience that the Bernese PAO in combination with appropriate adjunctive treatments has provided a very satisfactory surgical approach in the treatment of CP hip dysplasia. In the adolescent and young adult with spastic CP, utilizing the Bernese PAO technique makes it possible to obtain redirection of often a very severe acetabular dysplasia. Adjunctive soft tissue procedures and a proximal femoral osteotomy are frequently necessary to maintain postoperative stability. A notable improvement in the quality of life and function directly attributable to our surgical treatment of their pre-existing problematic hip dysplasia has been consistently noted in early follow-up for our patients. LEVEL OF EVIDENCE: Level IV-therapeutic.


Assuntos
Paralisia Cerebral/complicações , Fêmur/cirurgia , Luxação do Quadril/cirurgia , Osteotomia/métodos , Acetábulo/cirurgia , Adolescente , Adulto , Artralgia , Feminino , Luxação do Quadril/etiologia , Humanos , Masculino , Osteotomia/efeitos adversos , Qualidade de Vida , Radiografia , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
5.
Diabetologia ; 63(5): 1043-1054, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31974732

RESUMO

AIMS/HYPOTHESIS: Evidence that glucose-dependent insulinotropic peptide (GIP) and/or the GIP receptor (GIPR) are involved in cardiovascular biology is emerging. We hypothesised that GIP has untoward effects on cardiovascular biology, in contrast to glucagon-like peptide 1 (GLP-1), and therefore investigated the effects of GIP and GLP-1 concentrations on cardiovascular disease (CVD) and mortality risk. METHODS: GIP concentrations were successfully measured during OGTTs in two independent populations (Malmö Diet Cancer-Cardiovascular Cohort [MDC-CC] and Prevalence, Prediction and Prevention of Diabetes in Botnia [PPP-Botnia]) in a total of 8044 subjects. GLP-1 (n = 3625) was measured in MDC-CC. The incidence of CVD and mortality was assessed via national/regional registers or questionnaires. Further, a two-sample Mendelian randomisation (2SMR) analysis between the GIP pathway and outcomes (coronary artery disease [CAD] and myocardial infarction) was carried out using a GIP-associated genetic variant, rs1800437, as instrumental variable. An additional reverse 2SMR was performed with CAD as exposure variable and GIP as outcome variable, with the instrumental variables constructed from 114 known genetic risk variants for CAD. RESULTS: In meta-analyses, higher fasting levels of GIP were associated with risk of higher total mortality (HR[95% CI] = 1.22 [1.11, 1.35]; p = 4.5 × 10-5) and death from CVD (HR[95% CI] 1.30 [1.11, 1.52]; p = 0.001). In accordance, 2SMR analysis revealed that increasing GIP concentrations were associated with CAD and myocardial infarction, and an additional reverse 2SMR revealed no significant effect of CAD on GIP levels, thus confirming a possible effect solely of GIP on CAD. CONCLUSIONS/INTERPRETATION: In two prospective, community-based studies, elevated levels of GIP were associated with greater risk of all-cause and cardiovascular mortality within 5-9 years of follow-up, whereas GLP-1 levels were not associated with excess risk. Further studies are warranted to determine the cardiovascular effects of GIP per se.


Assuntos
Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/mortalidade , Polipeptídeo Inibidor Gástrico/metabolismo , Glucose/metabolismo , Adulto , Idoso , Feminino , Genótipo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores dos Hormônios Gastrointestinais/metabolismo
6.
Proc Natl Acad Sci U S A ; 113(2): 434-9, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26712011

RESUMO

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.


Assuntos
Ciclo-Oxigenase 2/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Transdução de Sinais , Transcrição Gênica , Animais , Ciclo-Oxigenase 2/metabolismo , Ciclosporina/farmacologia , Citocinas/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Vida Livre de Germes , Rim/efeitos dos fármacos , Rim/metabolismo , Lipopolissacarídeos/farmacologia , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos
7.
J Emerg Med ; 56(3): 241-247, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30679068

RESUMO

BACKGROUND: Musculoskeletal (MSK) infections can be difficult to diagnose in acute care settings. The utility of clinical decision tools for pediatric MSK infections in an emergency department has not been well studied. OBJECTIVE: Our aim was to evaluate the performance of a septic hip clinical decision rule (CDR) in the evaluation of pediatric musculoskeletal infections. METHODS: We performed a retrospective study of children evaluated for an MSK infection in our emergency department from 2014 to 2016. Data collection included demographics, discharge diagnoses, and clinical/laboratory predictors from the CDR. A χ2 analysis and Wilcoxon rank-sum tests compared patients with and without MSK infections. Logistic regression analysis examined the predictors for MSK infections. A receiver operating characteristic (ROC) curve was calculated to evaluate the performance of the predictors. RESULTS: Of 996 evaluations included in the final analysis, 109 (10.9%) had MSK infections. In a multivariable model, an adjusted odds ratio (OR) was significant for fever (OR 3.9, 95% confidence interval [CI] 2.4-6.4), refusal to bear weight/pseudoparalysis (OR 4.4, 95% CI 2.7-7.1), and C-reactive protein (CRP) > 2.0 mg/dL (OR 5.4, 95% CI 3.2-9.1). The probability of infection was 75.1% with five predictors present, 1.9% for zero predictors, and 5.1% if one predictor was present. An ROC curve showed an area under the curve of 0.82, indicating moderate accuracy. CONCLUSIONS: A septic hip CDR demonstrates a low predicted probability of an MSK infection with zero or one clinical predictor present and moderate predictability with all five predictors. Fever, refusal to bear weight/pseudoparalysis, and CRP > 2.0 mg/dL performed best and should alert providers to consider other MSK infections in addition to septic arthritis.


Assuntos
Artrite Infecciosa/diagnóstico , Quadril/microbiologia , Pediatria/instrumentação , Adolescente , Artrite Infecciosa/fisiopatologia , Proteína C-Reativa/análise , Criança , Pré-Escolar , Sistemas de Apoio a Decisões Clínicas/instrumentação , Sistemas de Apoio a Decisões Clínicas/normas , Serviço Hospitalar de Emergência/organização & administração , Feminino , Febre/etiologia , Quadril/fisiopatologia , Humanos , Lactente , Modelos Logísticos , Masculino , Razão de Chances , Pediatria/métodos , Pediatria/normas , Valor Preditivo dos Testes , Estudos Prospectivos , Curva ROC , Estudos Retrospectivos , Suporte de Carga
8.
Pediatr Emerg Care ; 35(9): 605-610, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28328692

RESUMO

OBJECTIVES: Care process models (CPMs) for certain conditions have improved clinical outcomes in children. This study describes the implementation and impact of a CPM for the evaluation of musculoskeletal infections in a pediatric emergency department (ED). METHODS: A retrospective pre-post intervention study was performed to analyze the impact of a musculoskeletal infection CPM. Patients were identified retrospectively through electronic order history for imaging of an extremity or joint and recommended laboratory tests. Clinical outcomes evaluated included hospital length of stay (LOS), time to magnetic resonance imaging (MRI), time to administration of antibiotics, hospital admission rate, and 30-day readmission rate. RESULTS: Musculoskeletal infection evaluations completed in the ED were reviewed from 1 year before implementation (n = 383) and 2 years after implementation (n = 1219) of the CPM. A significant improvement in the time to antibiotic administration for all patients (4.3 vs 3.7 hours, P < 0.05) and for patients with confirmed musculoskeletal infections (9.5 vs 4.9 hours, P < 0.05) was observed after the implementation of the CPM. The overall time to MRI (13.2 vs 10.3 hours, P = 0.29) and hospital LOS (4.7 vs 3.7 days, P = 0.11) were improved for all patients but were not statistically significant. The admission rate and 30-day readmission were similar before and after the implementation of the CPM. CONCLUSIONS: The implementation of a musculoskeletal infection CPM has standardized the approach to the evaluation and diagnosis of musculoskeletal infections resulting in a significant decrease in the time to administer antibiotics and a downward trend in time to MRI and hospital LOS.


Assuntos
Protocolos Clínicos/normas , Serviço Hospitalar de Emergência/organização & administração , Medicina Baseada em Evidências/métodos , Hospitais Pediátricos/organização & administração , Antibacterianos/uso terapêutico , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/tratamento farmacológico , Criança , Feminino , Humanos , Masculino , Osteomielite/diagnóstico , Osteomielite/tratamento farmacológico , Readmissão do Paciente/estatística & dados numéricos , Piomiosite/diagnóstico , Piomiosite/tratamento farmacológico , Estudos Retrospectivos , Tempo para o Tratamento
10.
Arterioscler Thromb Vasc Biol ; 33(10): 2316-24, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23950143

RESUMO

OBJECTIVE: Interleukin-19 (IL-19) is a putative Th2, anti-inflammatory interleukin. Its expression and potential role in atherogenesis are unknown. IL-19 is not detected in normal artery and is expressed to a greater degree in plaque from symptomatic versus asymptomatic patients, suggesting a compensatory counter-regulatory function. We tested whether IL-19 could reduce atherosclerosis in susceptible mice and identified plausible mechanisms. APPROACH AND RESULTS: LDLR(-/-) mice fed an atherogenic diet and injected with either 1.0 or 10.0 ng/g per day recombinant mouse IL-19 had significantly less plaque area in the aortic arch compared with controls (P<0.0001). Weight gain, cholesterol, and triglyceride levels were not significantly different. Gene expression in splenocytes from IL-19-treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, interferon-γ, interleukin-1ß, and interleukin-12ß and increased expression of GATA3 and FoxP3 mRNA. A greater percentage of lymphocytes were Th2 polarized in IL-19-treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated that IL-19-treated mice have significantly less macrophage infiltrate compared with controls (P<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured endothelial cells, vascular smooth muscle cells, and bone marrow-derived macrophages with IL-19 resulted in a significant decrease in chemokine mRNA and mRNA stability protein human antigen R. CONCLUSIONS: These data suggest that IL-19 is a potent inhibitor of experimental atherosclerosis, with diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. This may identify IL-19 as a novel therapeutic to limit vascular inflammation.


Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Interleucina-10/farmacologia , Idoso , Animais , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Biomarcadores/sangue , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Colesterol/sangue , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Fatores de Tempo , Triglicerídeos/sangue
11.
Nat Commun ; 15(1): 315, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38182580

RESUMO

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.


Assuntos
Proteínas de Choque Térmico , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ciclo Celular , Divisão Celular , Citosol , Agregados Proteicos , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Gastroenterology ; 143(5): 1352-1360.e7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22841788

RESUMO

BACKGROUND & AIMS: The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS: We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS: Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS: NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.


Assuntos
Células Acinares/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neutrófilos/fisiologia , Pancreatite/metabolismo , Tripsinogênio/metabolismo , Células Acinares/efeitos dos fármacos , Amilases/sangue , Amilases/efeitos dos fármacos , Animais , Aorta/metabolismo , Núcleo Celular/metabolismo , Quimiocina CXCL2/efeitos dos fármacos , Quimiocina CXCL2/metabolismo , Pulmão/metabolismo , Camundongos , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/efeitos dos fármacos , Fatores de Transcrição NFATC/genética , Neutrófilos/efeitos dos fármacos , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/patologia , Peroxidase/efeitos dos fármacos , Peroxidase/metabolismo , Pirazóis/farmacologia , Transdução de Sinais , Baço/metabolismo , Estatísticas não Paramétricas , Ácido Taurocólico , Tripsinogênio/efeitos dos fármacos
13.
Elife ; 112022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35801695

RESUMO

Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease.


Assuntos
Fosforilação Oxidativa , Superóxidos , Dano ao DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo
14.
Arterioscler Thromb Vasc Biol ; 30(2): 218-24, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19965778

RESUMO

OBJECTIVE: Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. METHODS AND RESULTS: An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice. CONCLUSIONS: These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/etiologia , Hiperglicemia/metabolismo , Músculo Liso Vascular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteopontina/metabolismo , Animais , Apirase/farmacologia , Artérias/metabolismo , Sítios de Ligação , Calcineurina/metabolismo , Inibidores de Calcineurina , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Teste de Tolerância a Glucose , Humanos , Hiperglicemia/complicações , Hiperglicemia/tratamento farmacológico , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/deficiência , Fatores de Transcrição NFATC/genética , Osteopontina/deficiência , Osteopontina/genética , Regiões Promotoras Genéticas , Pirazóis/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Ativação Transcricional , Transfecção , Uridina Trifosfato/metabolismo
15.
Cell Rep ; 35(13): 109328, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34192536

RESUMO

In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumulates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Genes Fúngicos , Células HeLa , Humanos , Mutação/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Ligação Proteica , Saccharomyces cerevisiae/genética
16.
Cell Rep ; 34(3): 108637, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33472077

RESUMO

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.


Assuntos
Núcleo Celular/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Transdução de Sinais
17.
Proteomics ; 10(11): 2066-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20514648

RESUMO

The Human Antibody Initiative (HAI) aims to promote and facilitate the use of antibodies for proteomics research. The 6th workshop for the HUPO Antibody Initiative (HAI) held in September 2009 was co-chaired by Michael Snyder and Mathias Uhlen and discussed several aspects of antibody production, their validation, and attempts to standardise this process, in particular, when subsequently described in the literature. An update on the progress of the Human Protein Atlas was also presented to the attendees.


Assuntos
Anticorpos , Proteômica/métodos , Canadá , Humanos
18.
Proteomics ; 10(6): 1141-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20175080

RESUMO

Membrane proteins are key molecules in the cell, and are important targets for pharmaceutical drugs. Few three-dimensional structures of membrane proteins have been obtained, which makes computational prediction of membrane proteins crucial for studies of these key molecules. Here, seven membrane protein topology prediction methods based on different underlying algorithms, such as hidden Markov models, neural networks and support vector machines, have been used for analysis of the protein sequences from the 21,416 annotated genes in the human genome. The number of genes coding for a protein with predicted alpha-helical transmembrane region(s) ranged from 5508 to 7651, depending on the method used. Based on a majority decision method, we estimate 5539 human genes to code for membrane proteins, corresponding to approximately 26% of the human protein-coding genes. The largest fraction of these proteins has only one predicted transmembrane region, but there are also many proteins with seven predicted transmembrane regions, including the G-protein coupled receptors. A visualization tool displaying the topologies suggested by the eight prediction methods, for all predicted membrane proteins, is available on the public Human Protein Atlas portal (www.proteinatlas.org).


Assuntos
Proteínas de Membrana/genética , Proteoma , Algoritmos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Genoma Humano , Humanos , Proteínas de Membrana/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
19.
Mol Syst Biol ; 5: 337, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20029370

RESUMO

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced.


Assuntos
Bases de Dados de Proteínas , Regulação da Expressão Gênica , Análise Serial de Proteínas , Proteômica/métodos , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Análise por Conglomerados , Genótipo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Fenótipo
20.
Arterioscler Thromb Vasc Biol ; 29(10): 1465-70, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19755528

RESUMO

OBJECTIVE: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. METHODS AND RESULTS: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. CONCLUSIONS: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.


Assuntos
Aterosclerose/etiologia , Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/etiologia , Inflamação/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Apolipoproteínas E/fisiologia , Autoanticorpos/análise , Glicemia/análise , Artérias Cerebrais/química , Lipoproteínas LDL/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Estreptozocina , Molécula 1 de Adesão de Célula Vascular/sangue
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