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1.
Toxicol Lett ; 320: 19-27, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778773

RESUMO

The deleterious effects of glucocorticoids on glucose homeostasis limit their clinical use. There is substantial evidence demonstrating that islet function impaired by long-term glucocorticoids exposure is a core defect in the progression of impaired glucose tolerance to diabetes. The activity of heat-shock protein (Hsp) 90 is required to maintain the hormone-binding activity and stability of glucocorticoid receptor (GR). In the present study, Hsp90 inhibition by 17-DMAG counteracted dexamethasone-mediated inhibition of glucose-stimulated insulin secretion in isolated rat islets as well as expressions of neuropeptide Y (NPY) and somatostatin receptor 3 (SSTR3), two negative regulators of insulin secretion. Like 17-DMAG, both the pan-histone deacetylase (HDAC) inhibitor TSA and HDAC6 inhibitor Tubacin exhibited a similar action in protecting islet function against dexamethasone-induced injury, along with the downregulation of NPY and SSTR3 expressions. The hyperacetylation of Hsp90 by TSA and Tubacin disrupted its binding ability to GR and blocked dexamethasone-elicited nuclear translocation of GR in INS-1 ß-cell lines. In addition, Tubacin treatment triggered the GR protein degradation through the ubiquitin-proteasome pathway. These findings suggest that Hsp90 acetylation by inhibiting HDAC6 activity may be a potential strategy to prevent the development of steroid diabetes mellitus via alleviating glucocorticoid-impaired islet function.


Assuntos
Anilidas/farmacologia , Benzoquinonas/farmacologia , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Acetilação , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Ratos Sprague-Dawley , Via Secretória , Técnicas de Cultura de Tecidos
2.
J Clin Pathol ; 73(1): 7-13, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31422373

RESUMO

AIMS: Hereditary protein S (PS) deficiency is one of the natural anticoagulant deficiencies causing thrombophilia. We herein described a young male with recurrent deep venous thrombosis, who was diagnosed as type I PS deficiency with compound heterozygous mutations of PROS1 gene. We aimed to analyse the relationship between the genotype and phenotype detection and investigate the pathological mechanisms of PROS1 mutations causing PS deficiency. METHODS: Genetic analysis of PROS1 gene was carried out by direct sequencing. Thrombin generation potential and the inhibition function of thrombin generation by plasma PS were detected by thrombin generation test (TGT). The mRNA transcription level of mutant PS in vitro was measured by real-time PCR, while the protein level was evaluated by western blot and ELISA. Cellular distribution of the protein was further analysed by immunofluorescence. RESULTS: Compound heterozygous mutations (PROS1 c.1551_1552delinsG, p.Thr518Argfs*39 and PROS1 c.1681C>T, p.Arg561Trp) were identified in the propositus, and the former one was a novel small indel mutation. TGT results showed impaired inhibition of thrombin generation with the addition of activated protein C in his parents with certain heterozygous mutations. In vitro expression study, p.Thr518Argfs*39 mutant produced truncated protein retained in the cytoplasm, while p.Arg561Trp mutant partially affected the secretion of PS. Both mutations are located in C-terminal sex hormone-binding globulin (SHBG)-like domain of PS. CONCLUSIONS: Compound heterozygous mutations identified in the study have strong detrimental effect, causing severe type I PS deficiency in the propositus. SHBG-like domain of PS might play an important role in PS secretion system.


Assuntos
Coagulação Sanguínea/genética , Proteínas Sanguíneas/genética , Heterozigoto , Mutação , Deficiência de Proteína S/genética , Trombose Venosa/genética , Adulto , Proteínas Sanguíneas/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Deficiência de Proteína S/sangue , Deficiência de Proteína S/diagnóstico , Recidiva , Via Secretória , Índice de Gravidade de Doença , Trombina/metabolismo , Trombose Venosa/sangue , Trombose Venosa/diagnóstico
3.
Mater Sci Eng C Mater Biol Appl ; 104: 109932, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499934

RESUMO

Nanomaterial based paints are in current demand in the area of surface protective coatings due to the significant advances made to improve their antibacterial and anticorrosion characteristics. In this work, we have developed magnetic graphene oxide (MGO) paint with the incorporation of cobalt ferrite (CF) and graphene oxide (GO) along with paint materials by using high energy ball milling (HEBM). Morphological, elemental and functional analysis of the MGO paint is studied with ESEM, AFM, Raman, FTIR spectroscopy. EDS and PIXE methods are used for elemental analysis. Thermal analysis shows that the MGO film was stable up to 100 °C. The saturation magnetization of CF MNP is observed as 76 emu/g and it is reduced to 12 emu/g for MGP paint. The detailed antibacterial study of the prepared MGO paint has performed with S. typhimurium and E. coli. The dead-live assessment shows the dead population for S. typhimurium is superior up to 82% whereas it is 20% for E. coli. The morphological damage of bacterial cells is studied using SEM technique. Flow cytometry analysis of reactive oxygen species (ROS) generation experiments and computational analysis supported the proposed mechanism of induced ROS for the damage of bacterial membrane via interaction of GO and CF with bacterial proteins leading to alteration in their functionality. The observed results indicate that the prepared MGO paint could be a better candidate in the area of nano paint for surface protective coatings.


Assuntos
Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/síntese química , Compostos Férricos/síntese química , Grafite/síntese química , Nanopartículas de Magnetita/química , Via Secretória/efeitos dos fármacos , Antibacterianos/síntese química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Materiais Revestidos Biocompatíveis/farmacologia , Cobalto/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Compostos Férricos/farmacologia , Grafite/farmacologia , Humanos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Propriedades de Superfície , Temperatura Ambiente , Termogravimetria , Vibração
4.
Nutrients ; 11(8)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382615

RESUMO

The liver plays a pivotal role in whole-body carbohydrate, lipid, and protein metabolism. One of the key regulators of glucose and lipid metabolism are hepatokines, which are found among the liver secreted proteins, defined as liver secretome. To elucidate the composition of the human liver secretome and identify hepatokines in primary human hepatocytes (PHH), we conducted comprehensive protein profiling on conditioned medium (CM) of PHH. Secretome profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) identified 691 potential hepatokines in PHH. Subsequently, pathway analysis assigned these proteins to acute phase response, coagulation, and complement system pathways. The secretome of PHH was compared to the secreted proteins of the liver hepatoma cell line HepG2. Although the secretome of PHH and HepG2 cells show a high overlap, the HepG2 secretome rather mirrors the fetal liver with some cancer characteristics. Collectively, our study represents one of the most comprehensive secretome profiling approaches for PHH, allowing new insights into the composition of the secretome derived from primary human material, and points out strength and weakness of using HepG2 cell secretome as a model for the analysis of the human liver secretome.


Assuntos
Hepatócitos/metabolismo , Proteínas/metabolismo , Cromatografia de Fase Reversa , Células Hep G2 , Humanos , Cultura Primária de Células , Proteômica/métodos , Via Secretória , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
5.
Int J Mol Sci ; 20(15)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349735

RESUMO

Radiation therapy, which applies high-energy rays, to eradicate tumor cells, is considered an essential therapy for the patients with breast cancer. Most tumor cells secrete exosomes, which are involved in cell-to-cell communication in tumor tissue and contribute therapeutic resistance and promote tumor aggressiveness. Here, we investigated the effect of clinically applicable doses of X-ray irradiation (2, 4, 6, 8, 10 Gy) on the dynamics of the exosomes' activity in MCF-7 breast cancer cells. Survival and apoptosis rate of cells against X-ray doses was examined using MTT and flow cytometry assays, respectively. Whereas, the levels of reactive oxygen species (ROS) in the X-ray-treated cells were detected by fluorometric method. The mRNA levels of vital genes involved in exosome biogenesis and secretion including Alix, Rab11, Rab27a, Rab27b, TSPA8, and CD63 were measured by real-time PCR. The protein level of CD63 was examined by Western blotting. Additionally, exosomes were characterized by monitoring acetylcholinesterase activity, transmission electron microscopy, size determination, and zeta potential. The result showed that in comparison with control group cell survival and the percentage of apoptotic cells as well as amount of ROS dose-dependently decreased and increased in irradiated cells respectively (p < 0.05). The expression level of genes including Alix, Rab27a, Rab27b, TSPA8, and CD63 as well as the protein level of CD63 upraised according to an increase in X-ray dose (p < 0.05). We found that concurrent with an increasing dose of X-ray, the acetylcholinesterase activity, size, and zeta-potential values of exosomes from irradiated cells increased (p < 0.05). Data suggest X-ray could activate exosome biogenesis and secretion in MCF-7 cells in a dose-dependent way, suggesting the therapeutic response of cells via ROS and exosome activity.


Assuntos
Neoplasias da Mama/metabolismo , Exossomos/metabolismo , Radiação Ionizante , Via Secretória/efeitos da radiação , Acetilcolinesterase/metabolismo , Apoptose/efeitos da radiação , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Comunicação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Células MCF-7 , Tolerância a Radiação , Espécies Reativas de Oxigênio
6.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261750

RESUMO

Acetylation of nuclear apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is associated with its extracellular secretion, despite the lack of an N-terminal protein secretion signal. In this study, we investigated plasma membrane targeting and translocation of APE1/Ref-1 in HEK293T cells with enhanced acetylation. While APE1/Ref-1 targeting was not affected by inhibition of the endoplasmic reticulum/Golgi-dependent secretion, its secretion was reduced by inhibitors of ATP-binding cassette (ABC) transporters, and siRNA-mediated down-regulation of ABC transporter A1. The association between APE1/Ref-1 and ABCA1 transporter was confirmed by proximal ligation assay and immunoprecipitation experiments. An APE1/Ref-1 construct with mutated acetylation sites (K6/K7R) showed reduced co-localization with ABC transporter A1. Exposure of trichostatin A (TSA) induced the acetylation of APE1/Ref-1, which translocated into membrane fraction. Taken together, acetylation of APE1/Ref-1 is considered to be necessary for its extracellular targeting via non-classical secretory pathway using the ABCA1 transporter.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Via Secretória , Acetilação , Motivos de Aminoácidos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico/efeitos dos fármacos
7.
Blood ; 134(12): 979-982, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31262780

RESUMO

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular hemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von Willebrand factor [VWF]). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this via a proximity proteomics approach employing the peroxidase APEX2 coupled with 2 known WPB-associated proteins: the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells, and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. Whereas these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteômica/métodos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Biotinilação , Células Cultivadas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Exocitose/fisiologia , Humanos , Enzimas Multifuncionais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via Secretória/fisiologia
8.
Gastroenterology ; 157(4): 1093-1108.e11, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31325428

RESUMO

BACKGROUND & AIMS: Inflammation, injury, and infection up-regulate expression of the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in the intestinal epithelium. We studied the effects of cell-specific IDO1 expression in the epithelium at baseline and during intestinal inflammation in mice. METHODS: We generated transgenic mice that overexpress fluorescence-tagged IDO1 in the intestinal epithelium under control of the villin promoter (IDO1-TG). We generated intestinal epithelial spheroids from mice with full-length Ido1 (controls), disruption of Ido1 (knockout mice), and IDO1-TG and analyzed them for stem cell and differentiation markers by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. Some mice were gavaged with enteropathogenic Escherichia coli (E2348/69) to induce infectious ileitis, and ileum contents were quantified by polymerase chain reaction. Separate sets of mice were given dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid to induce colitis; intestinal tissues were analyzed by histology. We utilized published data sets GSE75214 and GDS2642 of RNA expression data from ilea of healthy individuals undergoing screening colonoscopies (controls) and patients with Crohn's disease. RESULTS: Histologic analysis of small intestine tissues from IDO1-TG mice revealed increases in secretory cells. Enteroids derived from IDO1-TG intestine had increased markers of stem, goblet, Paneth, enteroendocrine, and tuft cells, compared with control enteroids, with a concomitant decrease in markers of absorptive cells. IDO1 interacted non-enzymatically with the aryl hydrocarbon receptor to inhibit activation of NOTCH1. Intestinal mucus layers from IDO1-TG mice were 2-fold thicker than mucus layers from control mice, with increased proportions of Akkermansia muciniphila and Mucispirillum schaedleri. Compared to controls, IDO1-TG mice demonstrated an 85% reduction in ileal bacteria (P = .03) when challenged with enteropathogenic E coli, and were protected from immune infiltration, crypt dropout, and ulcers following administration of dextran sodium sulfate or 2,4,6-trinitrobenzenesulfonic acid. In ilea of Crohn's disease patients, increased expression of IDO1 correlated with increased levels of MUC2, LYZ1, and aryl hydrocarbon receptor, but reduced levels of SLC2A5. CONCLUSIONS: In mice, expression of IDO1 in the intestinal epithelial promotes secretory cell differentiation and mucus production; levels of IDO1 are positively correlated with secretory cell markers in ilea of healthy individuals and Crohn's disease patients. We propose that IDO1 contributes to intestinal homeostasis.


Assuntos
Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Microbioma Gastrointestinal , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/microbiologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Notch/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Estudos de Casos e Controles , Linhagem Celular , Linhagem da Célula , Modelos Animais de Doenças , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Genótipo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/deficiência , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Camundongos Knockout , Fenótipo , Receptores de Hidrocarboneto Arílico/genética , Receptores Notch/genética , Via Secretória , Transdução de Sinais , Células-Tronco/enzimologia , Células-Tronco/microbiologia , Células-Tronco/patologia
9.
PLoS Pathog ; 15(7): e1007982, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356625

RESUMO

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Leishmania/fisiologia , Leishmania/patogenicidade , Proteínas de Protozoários/fisiologia , Fatores de Virulência/fisiologia , Animais , Retículo Endoplasmático/parasitologia , Feminino , Glicoesfingolipídeos/fisiologia , Humanos , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Metaloendopeptidases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fagócitos/parasitologia , Fagocitose , Fagossomos/parasitologia , Proteínas Qa-SNARE/fisiologia , Proteínas R-SNARE/fisiologia , Via Secretória , Vacúolos/parasitologia , Virulência
10.
Biochimie ; 166: 161-172, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31212040

RESUMO

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.


Assuntos
Precursores Enzimáticos/metabolismo , /metabolismo , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Processamento de Proteína Pós-Traducional , Via Secretória
11.
Glycobiology ; 29(8): 562-571, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31094418

RESUMO

ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62's PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools-nano-flow liquid chromatography with high-definition electrospray mass spectrometry-to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions.


Assuntos
Acanthocheilonema/metabolismo , Proteínas de Helminto/química , Processamento de Proteína Pós-Traducional , Proteoma/química , Glicosilação , Proteínas de Helminto/metabolismo , Proteoma/metabolismo , Via Secretória
12.
Cells ; 8(5)2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100966

RESUMO

Mesenchymal stem cell (MSC)-sourced secretome, defined as the set of MSC-derived bioactive factors (soluble proteins, nucleic acids, lipids and extracellular vesicles), showed therapeutic effects similar to those observed after transplantation of MSCs. MSC-derived secretome may bypass many side effects of MSC-based therapy, including unwanted differentiation of engrafted MSCs. In contrast to MSCs which had to be expanded in culture to reach optimal cell number for transplantation, MSC-sourced secretome is immediately available for treatment of acute conditions, including fulminant hepatitis, cerebral ischemia and myocardial infarction. Additionally, MSC-derived secretome could be massively produced from commercially available cell lines avoiding invasive cell collection procedure. In this review article we emphasized molecular and cellular mechanisms that were responsible for beneficial effects of MSC-derived secretomes in the treatment of degenerative and inflammatory diseases of hepatobiliary, respiratory, musculoskeletal, gastrointestinal, cardiovascular and nervous system. Results obtained in a large number of studies suggested that administration of MSC-derived secretomes represents a new, cell-free therapeutic approach for attenuation of inflammatory and degenerative diseases. Therapeutic effects of MSC-sourced secretomes relied on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of anti-apoptotic and pro-survival pathways that resulted in tissue repair and regeneration.


Assuntos
Imunomodulação , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Regeneração , Medicina Regenerativa/métodos , Via Secretória/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais , Camundongos
13.
Int J Mol Sci ; 20(10)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137607

RESUMO

Communication between cells is quintessential for biological function and cellular homeostasis. Membrane-bound extracellular vesicles known as exosomes play pivotal roles in mediating intercellular communication in tumor microenvironments. These vesicles and exosomes carry and transfer biomolecules such as proteins, lipids and nucleic acids. Here we focus on exosomes secreted from senescent cells. Cellular senescence can alter the microenvironment and influence neighbouring cells via the senescence-associated secretory phenotype (SASP), which consists of factors such as cytokines, chemokines, matrix proteases and growth factors. This review focuses on exosomes as emerging SASP components that can confer pro-tumorigenic effects in pre-malignant recipient cells. This is in addition to their role in carrying SASP factors. Transfer of such exosomal components may potentially lead to cell proliferation, inflammation and chromosomal instability, and consequently cancer initiation. Senescent cells are known to gather in various tissues with age; eliminating senescent cells or blocking the detrimental effects of the SASP has been shown to alleviate multiple age-related phenotypes. Hence, we speculate that a better understanding of the role of exosomes released from senescent cells in the context of cancer biology may have implications for elucidating mechanisms by which aging promotes cancer and other age-related diseases, and how therapeutic resistance is exacerbated with age.


Assuntos
Carcinogênese/metabolismo , Senescência Celular , Exossomos/metabolismo , Via Secretória , Animais , Humanos , Fenótipo
14.
Tuberculosis (Edinb) ; 115: 1-13, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30948163

RESUMO

Post-translational modifications represent a key aspect of enzyme and protein regulation and function. Post-translational modifications are involved in signaling and response to stress, adaptation to changing environments, regulation of toxic and damaged proteins, proteins localization and host-pathogen interactions. Glycosylation in Mycobacterium tuberculosis (Mtb), is a post-translational modification often found in conjunction with acylation in mycobacterial proteins. Since the discovery of glycosylated proteins in the early 1980's, important advances in our understanding of the mechanisms of protein glycosylation have been made. The number of known glycosylated substrates in Mtb has grown through the years, yet many questions remain. This review will explore the current knowledge on protein glycosylation in Mtb, causative agent of Tuberculosis and number one infectious killer in the world. The mechanism and significance of this post-translational modification, as well as maturation, export and acylation of glycosylated proteins will be reviewed. We expect to provide the reader with an overall view of protein glycosylation in Mtb, as well as the significance of this post-translational modification to the physiology and host-pathogen interactions of this important pathogen. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011081 and 10.6019/PXD011081.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Acilação/fisiologia , Glicoproteínas/metabolismo , Glicosilação , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Celular/fisiologia , Metabolismo dos Lipídeos/fisiologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Via Secretória/fisiologia , Transdução de Sinais/fisiologia , Tuberculose/enzimologia , Tuberculose/imunologia , Tuberculose/metabolismo
15.
Protein J ; 38(3): 317-329, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004255

RESUMO

The site of protein folding and maturation for the majority of proteins that are secreted, localized to the plasma membrane or targeted to endomembrane compartments is the endoplasmic reticulum (ER). It is essential that proteins targeted to the ER are properly folded in order to carry out their function, as well as maintain protein homeostasis, as accumulation of misfolded proteins could lead to the formation of cytotoxic aggregates. Because protein folding is an error-prone process, the ER contains protein quality control networks that act to optimize proper folding and trafficking of client proteins. If a protein is unable to reach its native state, it is targeted for ER retention and subsequent degradation. The protein quality control networks of the ER that oversee this evaluation or interrogation process that decides the fate of maturing nascent chains is comprised of three general types of families: the classical chaperones, the carbohydrate-dependent system, and the thiol-dependent system. The cooperative action of these families promotes protein quality control and protein homeostasis in the ER. This review will describe the families of the ER protein quality control network and discuss the functions of individual members.


Assuntos
Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Chaperonas Moleculares/metabolismo , Glicosilação , Dobramento de Proteína , Via Secretória
16.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30927348

RESUMO

Bacteria use a variety of mechanisms to translocate proteins from the cytoplasm, where they are synthesized, to the cell surface or extracellular environment or directly into other cells, where they perform their ultimate functions. Type V secretion systems (T5SS) use ß-barrel transporter domains to export passenger domains across the outer membranes of Gram-negative bacteria. Distinct among T5SS are type Vb or two-partner secretion (TPS) systems in which the transporter and passenger are separate proteins, necessitating a mechanism for passenger-translocator recognition in the periplasm and providing the potential for reuse of the translocator. This review describes current knowledge of the TPS translocation mechanism, using Bordetella filamentous hemagglutinin (FHA) and its transporter FhaC as a model. We present the hypothesis that the TPS pathway may be a general mechanism for contact-dependent delivery of toxins to target cells.


Assuntos
Bordetella/metabolismo , Hemaglutininas/metabolismo , Via Secretória/fisiologia , Adesinas Bacterianas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella/patogenicidade , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Bactérias Gram-Negativas , Proteínas de Membrana Transportadoras , Modelos Moleculares , Sistemas de Secreção Tipo V/metabolismo , Virulência , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/microbiologia
17.
Nat Rev Urol ; 16(6): 363-375, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30923338

RESUMO

The extensive arsenal of bioactive molecules secreted by mesenchymal stem cells (MSCs), known as the secretome, has demonstrated considerable therapeutic benefit in regenerative medicine. Investigation into the therapeutic potential of the secretome has enabled researchers to replicate the anti-inflammatory, pro-angiogenic and trophic effects of stem cells without the need for the cells themselves. Furthermore, treatment with the MSC secretome could circumvent hurdles associated with cellular therapy, including oncogenic transformation, immunoreactivity and cost. Thus, a clear rationale exists for investigating the therapeutic potential of the MSC secretome in regenerative urology. Indeed, preclinical studies have demonstrated the therapeutic benefits of the MSC secretome in models of stress urinary incontinence, renal disease, bladder dysfunction and erectile dysfunction. However, the specific mechanisms underpinning therapeutic activity are unclear and require further research before clinical translation. Improvements in current proteomic methods used to characterize the secretome will be necessary to provide further insight into stem cells and their secretome in regenerative urology.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Via Secretória , Vesículas Extracelulares , Humanos , Transplante de Células-Tronco Mesenquimais , Medicina Regenerativa/métodos , Procedimentos Cirúrgicos Urológicos/métodos , Urologia/métodos
18.
World J Microbiol Biotechnol ; 35(4): 54, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900052

RESUMO

Filamentous fungi are important microorganisms used in industrial production of proteins and enzymes. Among these organisms, Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability. In recent years, there have been tremendous achievements in understanding the molecular mechanisms of the secretory pathways in filamentous fungi. The acquired pieces of knowledge can be harnessed to enhance protein production in filamentous fungi with assistance of state-of-the-art genetic engineering techniques.


Assuntos
Proteínas Fúngicas/biossíntese , Fungos/metabolismo , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Aspergillus/metabolismo , Códon , Proteínas Fúngicas/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Glicosilação , Peptídeos/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Saccharomycetales/metabolismo , Via Secretória/genética , Trichoderma/metabolismo
19.
EcoSal Plus ; 8(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30892177

RESUMO

In 1989, Normark and coworkers reported on fibrous surface structures called curli on strains of Escherichia coli that were suspected of causing bovine mastitis. Subsequent work by many groups has revealed an elegant and highly regulated curli biogenesis pathway also referred to as the type VIII secretion system. Curli biogenesis is governed by two divergently transcribed operons, csgBAC and csgDEFG. The csgBAC operon encodes the structural subunits of curli, CsgA and CsgB, along with a chaperone-like protein, CsgC. The csgDEFG operon encodes the accessory proteins required for efficient transcription, secretion, and assembly of the curli fiber. CsgA and CsgB are secreted as largely unstructured proteins and transition to ß-rich structures that aggregate into regular fibers at the cell surface. Since both of these proteins have been shown to be amyloidogenic in nature, the correct spatiotemporal synthesis of the curli fiber is of paramount importance for proper functioning and viability. Gram-negative bacteria have evolved an elegant machinery for the safe handling, secretion, and extracellular assembly of these amyloidogenic proteins.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Bactérias/química , Proteínas de Bactérias/química , Via Secretória , Escherichia coli/química , Proteínas de Escherichia coli/química , Biogênese de Organelas
20.
FEMS Yeast Res ; 19(3)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865773

RESUMO

Although there are similarities in the core steps of the secretion pathway from yeast to higher eukaryotes, significant functional differences exist even among diverse yeast species. Here, we used next-generation sequencing to identify two mutations in the Kluyveromyces lactis KlSEC59 gene, encoding dolichol kinase (DK), which are responsible for an enhanced secretion phenotype in a previously isolated mutant, MD2/1-9. Compared with the temperature-sensitive Saccharomyces cerevisiae sec59-1 mutant, which exhibits reduced N-glycosylation and decreased secretory efficacy, the identified K. lactis DK mutations had fewer effects on glycosylation, as well as on survival at high temperature and cell wall integrity. Moreover, despite some glycosylation defects, double DK mutations (G405S and I419S) in the K. lactis mutant strain demonstrated three times the level of recombinant α-amylase secretion as the wild-type strain. Overexpression of potential suppressors KlMNN10, KlSEL1, KlERG20, KlSRT1, KlRER2, KlCAX4, KlLPP1 and KlDPP1 in the DK-mutant strain restored carboxypeptidase Y glycosylation to different extents and, with the exception of KISRT1, reduced α-amylase secretion to levels observed in wild-type cells. Our results suggest that enhanced secretion related to reduced activity of mutant DK in K. lactis results from mild glycosylation changes that affect activity of other proteins in the secretory pathway.


Assuntos
Proteínas Fúngicas/genética , Kluyveromyces/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/biossíntese , Carboxipeptidases/metabolismo , Glicosilação , Sequenciamento de Nucleotídeos em Larga Escala , Kluyveromyces/enzimologia , Fenótipo , Via Secretória , alfa-Amilases/biossíntese
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