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2.
Biomed Pharmacother ; 134: 111174, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360046

RESUMO

In the last years, clusterin, a challenging and paradoxical apolipoprotein, has been of growing interest amongst a rising number of scientists. This enigmatic protein is present in all fluids of the organism besides within the intracellular matrix, and it plays diverse, and at times contrary, roles in a growing number of pathologies. It seems to vary its location and function to assure cellular survival being cytoprotective hence its significance in neuroprotection and cancer along with chemotherapy resistance. However, it can also lead to cellular arrest and its modulation to apoptosis. Additionally, it has been described to modulate pain, as well as linked to inflammation, cardioprotection, satiety and hunger, and possibly to addictive behaviour development. Thus, it has been postulated to be used both as a biomarker and a very explorable new therapeutic target for several conditions.


Assuntos
Clusterina/metabolismo , Transdução de Sinais , Animais , Clusterina/genética , Regulação da Expressão Gênica , Humanos , Via Secretória
4.
Nat Commun ; 11(1): 5189, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060596

RESUMO

Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn-/- cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations - one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.


Assuntos
Autofagossomos/metabolismo , Autofagossomos/virologia , Flavivirus/metabolismo , Quinases da Família src/metabolismo , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Chlorocebus aethiops , Dengue , Vírus da Dengue/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas SNARE/metabolismo , Via Secretória , Células Vero , Vírion/metabolismo , Liberação de Vírus , Zika virus/metabolismo , Infecção por Zika virus , Proteínas rab de Ligação ao GTP/metabolismo , Quinases da Família src/genética
5.
Int J Mol Sci ; 21(18)2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32957696

RESUMO

At present, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has quickly become a health emergency because no specifics vaccines or drugs, at this moment, are available. Recent studies have shown that the transplantation of mesenchymal stem cells (MSCs) into Coronavirus Disease 2019 (COVID-19) patients could represent a promising strategy for the development of new therapeutic methods. We speculate and suggest that the secretome of human Oral Tissue Stem Cells (hOTSCs), for their immunomodulatory and anti-inflammatory specific properties, could exert beneficial effects on the COVID-19 patients through an innovative aerosolisation technique. This non-invasive technique can offer multiple advantages in prophylaxis, as well as the prevention and treatment of severe epidemic respiratory syndrome with minimum risk and optimal therapeutic effects. This has the potential to create a novel pathway towards immunomodulatory therapy for the treatment of COVID-19 positive patients.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Células-Tronco Mesenquimais/metabolismo , Mucosa Bucal/citologia , Pneumonia Viral/tratamento farmacológico , Proteoma/uso terapêutico , Humanos , Fatores Imunológicos/metabolismo , Pandemias , Proteoma/metabolismo , Via Secretória
6.
Am J Physiol Renal Physiol ; 319(4): F664-F673, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32715764

RESUMO

Tubular changes contribute to the development of renal pathologies in diabetic kidney disease (DKD), including interstitial fibrosis. It is unclear how tubular cells relay signals to interstitial fibroblasts. Recently, exosomes have been recognized as crucial mediators of intercellular communication. We hypothesized that exosomes secreted from tubular cells may stimulate fibroblasts for interstitial fibrosis in DKD. In this study, we isolated and purified exosomes from the renal cortex of DKD mice and high glucose-treated mouse proximal tubular cells. Compared with nondiabetic mice, exosome secretion in kidney tissues decreased in DKD mice. Likewise, high glucose incubation reduced exosome secretion in mouse kidney proximal tubular BUMPT cells. To study the effect of tubular cell exosomes on fibroblasts, exosomes from BUMPT cells were added to renal fibroblast NRK-49F cell cultures. Notably, exosomes from high glucose conditioned BUMPT cells induced higher proliferation, significant morphological change, and substantial production of fibronectin, α-smooth muscle actin, and collagen type Ι in NRK-49F fibroblasts. Proteomics analysis was further performed to profile the proteins within tubular cell exosomes. Interestingly, 22 proteins were found to be differentially expressed between tubular exosomes derived from high glucose conditioned cells and those from normal glucose conditioned cells. Cytoscape analysis suggested the existence of two protein-protein interaction networks in these exosomal differentially expressed proteins. While one of the protein-protein interaction networks comprised enolase 1 (Eno1), heat shock protein family A member 8 (Hspa8), thioredoxin 1 (Txn1), peptidylprolyl isomerase A (Ppia), phosphoglycerate kinase 1 (Pgk1), DNA topoisomerase II-ß (Top2b), and ß-actin (Actb), the other had the family proteins of human leucocyte antigen F (Ywhag), a component of the ND10 nuclear body (Ywhae), interferon regulatory factor-8 (Ywhaq), and human leucocyte antigen A (Ywhaz). Gene expression analysis via Nephroseq showed a correlation of Eno1 expression with DKD clinical manifestation. In conclusion, DKD is associated with a decrease in exosome secretion in renal tubular cells. Exosomes from high glucose conditioned tubular cells may regulate the proliferation and activation of fibroblasts, contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.


Assuntos
Proliferação de Células , Nefropatias Diabéticas/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , Túbulos Renais Proximais/metabolismo , Comunicação Parácrina , Animais , Linhagem Celular , Técnicas de Cocultura , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Exossomos/genética , Exossomos/patologia , Fibroblastos/patologia , Fibrose , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Fosfopiruvato Hidratase/metabolismo , Mapas de Interação de Proteínas , Via Secretória , Transdução de Sinais
7.
Diab Vasc Dis Res ; 17(4): 1479164120945675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722929

RESUMO

Activation of the prostaglandin E2 receptor EP4 alters polarization of adipose tissue macrophages towards the anti-inflammatory M2 phenotype to suppress chronic inflammation. However, the role of EP4 signalling in pancreatic macrophages that affect insulin secretion is unclear. We examined the role of EP4 signalling in islet inflammation in vitro and in vivo. Obese diabetic db/db mice were treated with an EP4-selective agonist or vehicle for 4 weeks. Islet morphology did not significantly differ and glucose-stimulated insulin secretion was increased, whereas the pancreatic M1/M2 ratio was decreased in the EP4 agonist-treated group compared to the vehicle group. Because EP4 activation in MIN6 cells did not affect insulin secretion, we used a MIN6/macrophage co-culture system to evaluate the role of EP4 signalling in islet inflammation and subsequent inhibition of insulin release. Co-culture with M1-polarized macrophages markedly suppressed insulin expression in MIN6 cells; however, modulation of M1 polarization by the EP4 agonist significantly reversed the negative impact of co-cultivation on insulin production. The enhanced expression levels of pro-inflammatory cytokines in co-cultured MIN6 cells were markedly inhibited by EP4 agonist treatment of M1 macrophages. Thus, EP4 activation may suppress islet inflammation and protect ß-cell function by altering inflammatory macrophages in the diabetic pancreas.


Assuntos
Plasticidade Celular , Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Células Secretoras de Insulina/metabolismo , Macrófagos Peritoneais/metabolismo , Obesidade/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ativação de Macrófagos , Macrófagos Peritoneais/patologia , Camundongos , Obesidade/patologia , Fenótipo , Via Secretória , Transdução de Sinais
8.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32725137

RESUMO

Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway-mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology that could also be targeted with therapeutic objectives. Finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Via Secretória/fisiologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Via Secretória/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
9.
Arterioscler Thromb Vasc Biol ; 40(9): 2054-2069, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640907

RESUMO

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.


Assuntos
Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Catepsinas/biossíntese , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catepsinas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Exocitose , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout para ApoE , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , Via Secretória , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
10.
Diab Vasc Dis Res ; 17(3): 1479164120920582, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32506946

RESUMO

Obesity-related euglycaemic insulin resistance clusters with cardiometabolic risk factors, contributing to the development of both type 2 diabetes and cardiovascular disease. An increased thrombotic tendency in diabetes stems from platelet hyperactivity, enhanced activity of prothrombotic coagulation factors and impaired fibrinolysis. Furthermore, a low-grade inflammatory response and increased oxidative stress accelerate the atherosclerotic process and, together with an enhanced thrombotic environment, result in premature and more severe cardiovascular disease. The disruption of circadian cycles in man secondary to chronic obesity and loss of circadian cues is implicated in the increased risk of developing diabetes and cardiovascular disease. Levels of melatonin, the endogenous synchronizer of circadian rhythm, are reduced in individuals with vascular disease and those with deranged glucose metabolism. The anti-inflammatory, antihypertensive, antioxidative and antithrombotic activities of melatonin make it a potential therapeutic agent to reduce the risk of vascular occlusive disease in diabetes. The mechanisms behind melatonin-associated reduction in procoagulant response are not fully known. Current evidence suggests that melatonin inhibits platelet aggregation and might affect the coagulation cascade, altering fibrin clot structure and/or resistance to fibrinolysis. Large-scale clinical trials are warranted to investigate the effects of modulating the circadian clock on insulin resistance, glycaemia and cardiovascular outcome.


Assuntos
Aterosclerose/sangue , Ritmo Circadiano , Diabetes Mellitus Tipo 2/sangue , Melatonina/sangue , Trombose/sangue , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/etiologia , Aterosclerose/fisiopatologia , Coagulação Sanguínea , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Fibrinólise , Humanos , Resistência à Insulina , Melatonina/deficiência , Melatonina/uso terapêutico , Via Secretória , Transdução de Sinais , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/fisiopatologia
11.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G74-G86, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538138

RESUMO

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.


Assuntos
Células Acinares/metabolismo , Movimento Celular/fisiologia , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Amilases/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Glândula Parótida/metabolismo
12.
Ann Biol Clin (Paris) ; 78(3): 231-242, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32540812

RESUMO

The identification of leptin allowed the discovery of a new endocrine system. This major adipokine controlling energy homeostasis is also involved in the regulation of neuroendocrine function and fertility. Unfortunately, leptin is not able to treat common obesity, which associates hyperleptinemia and resistance to the hormone. Conversely, treatment with recombinant leptin is effective in situations of leptin deficiency. Several pathophysiological situations associated with adipose tissue dysfunctions and abnormal regulation of leptin secretion are discussed in this review. The advantage of the potential use of the leptin assay in some pathophysiological conditions is proposed.


Assuntos
Leptina/fisiologia , Adipocinas/fisiologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiopatologia , Animais , Homeostase/fisiologia , Humanos , Obesidade/metabolismo , Obesidade/fisiopatologia , Via Secretória/fisiologia
13.
Nat Commun ; 11(1): 2170, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358503

RESUMO

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Resistência à Doença/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Raízes de Plantas/metabolismo , Transcriptoma/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Parede Celular/química , Parede Celular/metabolismo , Endossomos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Plasmodioforídeos/patogenicidade , Via Secretória/genética , Solo , Proteínas de Transporte Vesicular/metabolismo
14.
Ann Hematol ; 99(7): 1531-1542, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32430703

RESUMO

In haemophilia, thrombin generation and fibrin deposition upon vascular injury critically depend on the tissue factor (TF)-driven coagulation pathway. TF expression by monocytes/macrophages and circulating microvesicles contributes to haemostasis, thrombosis and inflammation. Inflammation is a hallmark of blood-induced joint disease. The aim of this study is to correlate TF production by whole-blood monocytes with inflammatory markers and clinical parameters in patients with moderate-to-severe haemophilia A or B (n = 43) in comparison to healthy males (n = 23). Monocyte TF antigen and microvesicle-associated TF procoagulant activity (MV TF PCA) were measured immediately after blood draw (baseline) and following incubation of whole blood with buffer or lipopolysaccharide (LPS) using two-colour flow cytometry and chromogenic FXa generation assay, respectively. Patients with HIV or uncontrolled HBV/HCV infections were excluded. TF was hardly detectable and not different in baseline and buffer-treaded samples from both groups. Stimulation with LPS, however, induced monocyte TF production, with increased TF-specific mean fluorescence intensity (P = 0.08) and MV TF PCA (P < 0.05) in patients compared to controls. Patients also had elevated hs-CRP and IL-6 serum levels (P < 0.001), which correlated with LPS-induced TF parameters. Further exploratory analyses revealed that the presence of systemic (low-grade) inflammation and boosted LPS-induced monocyte TF production were mainly restricted to patients with clinically controlled HBV and/or HCV infection (n = 16), who were older and also had a significantly worse orthopaedic joint score than patients with no history of viral hepatitis (P < 0.01). Our study delineates a previously unrecognised link between systemic inflammation and inducible monocyte TF production in patients with haemophilia A or B.


Assuntos
Hemofilia A/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Tromboplastina/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Hemofilia A/patologia , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Via Secretória/efeitos dos fármacos , Índice de Gravidade de Doença , Adulto Jovem
15.
Am J Physiol Cell Physiol ; 318(6): C1284-C1293, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32320287

RESUMO

The present study aimed to elucidate the mechanisms by which leucine impacts the secretion of pancreatic enzymes, especially amylase, by studying the proteomics profiles of pancreatic acinar (PA) cells from dairy cows. PA cells, the experimental model, were treated with four concentrations of leucine (0, 0.23, 0.45, and 0.90 mM). The abundance of different proteins in the four leucine treatment groups was detected. Label-free proteomic analysis enabled the identification of 1,906 proteins in all four treatment groups, and 1,350 of these proteins showed common expression across the groups. The primary effects of leucine supplementation were increased (P < 0.05) citrate synthase and ATPase activity, which enlarged the cytosolic ATP pool, and the upregulation of secretory protein 61 (Sec61) expression, which promoted protein secretion. In summary, these results suggest that leucine increases citrate synthase in the TCA cycle and ATPase activity and promotes the Sec signaling pathway to increase the exocrine function of PA cells.


Assuntos
Células Acinares/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Leucina/farmacologia , Pâncreas Exócrino/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , alfa-Amilases/metabolismo , Células Acinares/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Bovinos , Células Cultivadas , Citrato (si)-Sintase/metabolismo , Indústria de Laticínios , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pâncreas Exócrino/enzimologia , Proteômica , Canais de Translocação SEC/metabolismo
16.
Cancer Genomics Proteomics ; 17(3): 225-236, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32345664

RESUMO

BACKGROUND: Malignant pleural mesothelioma (MPM) a rare neoplasm linked to asbestos exposure is characterized by a poor prognosis. Soluble mesothelin is currently considered the most specific diagnostic biomarker. The aim of the study was to identify novel biomarkers by proteomic analysis of two MPM cell lines secretome. MATERIALS AND METHODS: The protein patterns of MPM cells secretome were examined and compared to a non-malignant mesothelial cell line using two-dimensional gel electrophoresis coupled to mass spectrometry. Serum levels of candidate biomarkers were determined in MPM patients and control subjects. RESULTS: Two up-regulated proteins involved in cancer biology, prosaposin and quiescin Q6 sulfhydryl oxidase 1, were considered candidate biomarkers. Serum levels of both proteins were significantly higher in MPM patients than control subjects. Combining the data of each receiver-operating characteristic analysis predicted a good diagnostic accuracy. CONCLUSION: A panel of the putative biomarkers represents a promising tool for MPM diagnosis.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Pulmonares/sangue , Mesotelioma/sangue , Neoplasias Pleurais/sangue , Proteoma/metabolismo , Idoso , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/sangue , Humanos , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/patologia , Pessoa de Meia-Idade , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/sangue , Neoplasias Pleurais/patologia , Curva ROC , Saposinas/sangue , Via Secretória
17.
J Biosci Bioeng ; 130(2): 142-148, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32327386

RESUMO

As a large group of natural product with significant biological activities, triterpenoid secretion is of particular importance towards its bioproduction. Due to the lack of specific transporters, most triterpenoids are naturally accumulated inside the cells. In this study, by taking an antitumor triterpenoid ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid (GA-HLDOA) as example, we discovered that addition of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) or 2,6-dimethyl-ß-cyclodextrin (DM-ß-CD) enable the fast and sufficient secretion of GA-HLDOA by the recombinant Saccharomyces cerevisiae strain as constructed in our previous study. In addition, these cyclodextrins (CDs) could not enter into cells, while no significant change of the cell membrane fluidity was observed after CDs treatment. This discovery provides a potential generally applicable method for triterpenoid secretion.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Via Secretória/efeitos dos fármacos , Triterpenos/metabolismo , beta-Ciclodextrinas/farmacologia , Ciclodextrinas/farmacologia , Microbiologia Industrial , Saccharomyces cerevisiae/metabolismo
18.
Biochem Biophys Res Commun ; 526(4): 1036-1041, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32305137

RESUMO

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Genes de Plantas , Lipase/genética , Pólen/fisiologia , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Sequência Conservada/genética , Fertilidade/fisiologia , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Lipase/metabolismo , Filogenia , Infertilidade das Plantas/genética , Pólen/ultraestrutura , Via Secretória
19.
Nat Commun ; 11(1): 1780, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286267

RESUMO

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Concentração Inibidora 50 , Espectrometria de Massas , Proteínas de Protozoários/metabolismo , Pirazóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Via Secretória/efeitos dos fármacos
20.
Am J Physiol Cell Physiol ; 318(6): C1107-C1122, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267718

RESUMO

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Túbulos de Malpighi/metabolismo , Tetraspaninas/metabolismo , Junções Íntimas/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Impedância Elétrica , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Túbulos de Malpighi/embriologia , Túbulos de Malpighi/ultraestrutura , Via Secretória , Transdução de Sinais , Tetraspaninas/genética , Junções Íntimas/genética , Junções Íntimas/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
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