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1.
J Biol Regul Homeost Agents ; 34(4 Suppl. 2): 71-77, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33000604

RESUMO

Mucolipidosis II and III are lysosomal storage diseases caused by pathogenetic mutations in GNPTAB and GNPTG genes which cause an impaired activity of the lysosomal hydrolase N-acetylglucosamine- 1-phosphotransferase, a key enzyme in the synthesis of the mannose-6-phosphate targeting signals on lysosomal enzymes. Patients with MLII alpha/beta present coarse facial features, cessation of statural growth, important skeletal manifestations, impaired neuromotor development and cardiorespiratory involvement. All children appear to have cardiac involvement, but severe dilated cardiomyopathy is uncommon. In this report we describe the case of an 11-month-old girl who is affected by a MLII. Analysis of the GNPTAB gene identified at a heterozygous level the previously described gene variants c. 2693delA p(Lys898Serfs*13) and c. 2956C>T p(Arg986Cys). Her main clinical features were coarse face with gingival hypertrophy, dysostosis multiplex, recurrent respiratory infection and an early onset of dilated cardiomyopathy, an uncommon feature for MLII. To our knowledge, dilated cardiomyopathy has been previously described in literature in only two cases of MLII and in one patient affected by MLIII.


Assuntos
Cardiomiopatia Dilatada , Diabetes Mellitus Tipo 2 , Mucolipidoses , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/genética , Criança , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Lactente , Mucolipidoses/complicações , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , Transferases (Outros Grupos de Fosfato Substituídos)/genética
3.
J Biol Chem ; 295(10): 2932-2947, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31980461

RESUMO

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.


Assuntos
Diacilglicerol Quinase/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Alinhamento de Sequência , Motivo Estéril alfa , Espectrometria de Massas em Tandem , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética
4.
Am J Respir Cell Mol Biol ; 62(3): 342-353, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31517509

RESUMO

Sphingomyelin synthase is responsible for the production of sphingomyelin (SGM), the second most abundant phospholipid in mammalian plasma, from ceramide, a major sphingolipid. Knowledge of the effects of cigarette smoke on SGM production is limited. In the present study, we examined the effect of chronic cigarette smoke on sphingomyelin synthase (SGMS) activity and evaluated how the deficiency of Sgms2, one of the two isoforms of mammalian SGMS, impacts pulmonary function. Sgms2-knockout and wild-type control mice were exposed to cigarette smoke for 6 months, and pulmonary function testing was performed. SGMS2-dependent signaling was investigated in these mice and in human monocyte-derived macrophages of nonsmokers and human bronchial epithelial (HBE) cells isolated from healthy nonsmokers and subjects with chronic obstructive pulmonary disease (COPD). Chronic cigarette smoke reduces SGMS activity and Sgms2 gene expression in mouse lungs. Sgms2-deficient mice exhibited enhanced airway and tissue resistance after chronic cigarette smoke exposure, but had similar degrees of emphysema, compared with smoke-exposed wild-type mice. Sgms2-/- mice had greater AKT phosphorylation, peribronchial collagen deposition, and protease activity in their lungs after smoke inhalation. Similarly, we identified reduced SGMS2 expression and enhanced phosphorylation of AKT and protease production in HBE cells isolated from subjects with COPD. Selective inhibition of AKT activity or overexpression of SGMS2 reduced the production of several matrix metalloproteinases in HBE cells and monocyte-derived macrophages. Our study demonstrates that smoke-regulated Sgms2 gene expression influences key COPD features in mice, including airway resistance, AKT signaling, and protease production.


Assuntos
Resistência das Vias Respiratórias/fisiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Tabaco/efeitos adversos , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Animais , Brônquios/citologia , Células Cultivadas , Ceramidas/metabolismo , Células Epiteliais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Metaloproteinases da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologia
5.
FEBS Lett ; 594(3): 519-529, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31596951

RESUMO

The obligate intracellular bacterium Chlamydia trachomatis proliferates in the membranous compartment inclusion formed in host cells. The host ceramide transport protein CERT delivers ceramide from the endoplasmic reticulum to the Golgi complex for the synthesis of sphingomyelin (SM). Chlamydia trachomatis has been suggested to employ CERT to produce SM in the inclusion by host SM synthases (SMSs). Here, we found that C. trachomatis proliferates and produces infective progeny even in SMS1 and SMS2 double-knockout HeLa cells, but not in the SMS1/SMS2/CERT triple-knockout cells. Interestingly, infected cells convert ceramide to SM without host SMSs. These results suggest that C. trachomatis-infected cells can convert ceramide to SM without host SMSs after CERT-mediated transfer of ceramide to the inclusions.


Assuntos
Ceramidas/metabolismo , Chlamydia trachomatis/fisiologia , Esfingomielinas/metabolismo , Sequência de Bases , Técnicas de Inativação de Genes , Células HeLa , Humanos , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-31678513

RESUMO

Bacterial lipopolysaccharide (LPS) is recognized by CD14 protein and the Toll-like receptor (TLR)4/MD2 complex localized in the plasma membrane of immune cells. TLR4 triggers two signaling pathways engaging the MyD88 and TRIF adaptor proteins which lead to production of various pro-inflammatory cytokines. These processes are likely to be modulated by sphingomyelin, as the CD14 - TLR4 interaction takes place in plasma membrane rafts enriched in this lipid. To verify this assumption, we analyzed the influence of tricyclodecane-9-yl xanthogenate (D609), which was proven here to be an SMS inhibitor, and silencing of sphingomyelin synthase (SMS) 1 and/or SMS2 on LPS-induced signaling in macrophages. LPS up-regulated the expression and activity of SMS while exposure to D609 or silencing of SMS1 and SMS2 counteracted this action and led (except for SMS2 silencing) to a depletion of sphingomyelin in cells. Concomitantly, the MyD88- and TRIF-dependent signaling pathways of TLR4 were inhibited with the latter being especially sensitive to the reduction of the SMS1 and/or SMS2 activity. The D609 treatment and SMS1 and/or SMS2 depletion all reduced the level of CD14 protein in cells, which likely was an important determinant of the reduction of the LPS-induced pro-inflammatory responses.


Assuntos
Transdução de Sinais/imunologia , Esfingomielinas/metabolismo , Receptor 4 Toll-Like/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Norbornanos , Cultura Primária de Células , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos , Tionas/farmacologia , Receptor 4 Toll-Like/genética , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-31672571

RESUMO

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron­sulfur (FeS) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO4) and hydrogen peroxide (H2O2), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.


Assuntos
Cardiolipinas/metabolismo , Glutationa/biossíntese , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cisteína/metabolismo , Compostos Ferrosos/metabolismo , Ácido Glutâmico/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
8.
Zhonghua Er Ke Za Zhi ; 57(12): 950-954, 2019 Dec 02.
Artigo em Chinês | MEDLINE | ID: mdl-31795562

RESUMO

Objective: To investigate the clinical and genetic characteristics of 3 patients with mucolipidosis and to perform literature review. Methods: A retrospective analysis was made on the clinical data and genetic test results of 3 pedigrees with mucolipidosis. The patients were followed up at the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from February 2016 to August 2018. A neonatal inherited metabolic diseases gene panel including GNPTAB, GNPTG, MCOLN1, etc. was used for next-generation sequencing (NGS) based testing. Sanger sequencing was subsequently used to confirm the suspected pathological variants in the patients and their family members. Original papers on mucolipidosis published up to December 2018 were retrieved from PubMed, CNKI and WanFang databases by using the key words "mucolipidosis" AND "Chinese" . Results: The onset ages ranged from (9-90) days. The common clinical characteristics of the 3 patients are developmental delay and skeletal abnormalities. Targeted NGS revealed 5 different variations all in GNPTAB including p.Arg364Ter, p.Ser385Leu, p.Try404Ter, p. Arg587Ter, c.1284+1G>T. Two variants p.Ser385Leu and c.1284+1G>T were novel. Twenty-six cases of mucolipidosis have been reported in Chinese from 8 papers, which included 11 type ML Ⅱα/ß, 11 type ML Ⅲ α/ß and 4 type ML Ⅲ γ. c.2715+1G>A and p.Arg364Ter variants are likely the hot variants in Chinese ML patients. Conclusions: Mucolipidosis is a rare autosomal recessive disorder characterized by developmental delay and skeletal abnormalities. NGS plus Sanger sequencing detection is effective and accurate for making genetic diagnosis. p.Ser385Leu and c.1284+1G>T of GNPTAB gene are identified as novel pathogenic variants. GNPTAB gene is the main disease causing gene among Chinese ML patients, and c.2715+1G>A and p.Arg364Ter are the most common variants.


Assuntos
Mucolipidoses/diagnóstico , Mucolipidoses/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Feminino , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Mutação , Linhagem , Gravidez , Estudos Retrospectivos
9.
Mol Med ; 25(1): 56, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847800

RESUMO

BACKGROUND: There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. METHODS: We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2-/- mouse). RESULTS: When we compared Sp7-Cre;SMS1f/f;SMS2-/- mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2-/-) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/- and Sp7-Cre;SMS1+/+;SMS2-/-), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2-/- mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/f SMS2-/- mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. CONCLUSIONS: These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.


Assuntos
Diferenciação Celular/genética , Osteoblastos/fisiologia , Osteogênese/genética , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
10.
J Pediatr Endocrinol Metab ; 32(12): 1399-1402, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31758855

RESUMO

Background Mucolipidosis II α/ß (ML II) is an autosomal recessive disease associated with the abnormality of lysosomal enzyme trafficking. Case presentation We present an unusual patient with: (a) marked skeletal anomalies with secondary hyperparathyroidism; (b) serum intact parathyroid hormone level normalized by 7 weeks but abnormally elevated serum alkaline phosphate persisted; and (c) two mutations identified in the GNPTAB gene. One mutation, c.3503_3504delTC, is the most common mutation in ML II. However, the second mutation, c.2896delA, is a rare mutation for which clinical presentation has not been described previously.


Assuntos
Hiperparatireoidismo Secundário/patologia , Mucolipidoses/patologia , Adulto , Feminino , Idade Gestacional , Humanos , Hiperparatireoidismo Secundário/complicações , Hiperparatireoidismo Secundário/genética , Recém-Nascido , Mucolipidoses/complicações , Mucolipidoses/genética , Mutação , Prognóstico , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Adulto Jovem
11.
Eur Rev Med Pharmacol Sci ; 23(18): 8158-8167, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31599445

RESUMO

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) has been proven to be the most common liver disease in the world, which is a sterile liver disease and is characterized by chronic hepatic steatosis and inflammation. The first step of the spectrum of the disease is the non-alcoholic fatty liver (NAFL). Based on hepatocellular necrosis and inflammation, NAFL will progress to non-alcoholic steatohepatitis (NASH), which may have the potential to progress cirrhosis, and even hepatocellular carcinoma (HCC) in a few years. Kupffer cells (KCs) are liver-resident macrophages and have been proven to play a crucial role in NAFLD development. Cardiolipin is reported to be effective to trigger the activation of NLRP3 inflammasome through a ROS-independent signaling pathway. However, the exact mechanism of NLRP3 inflammasome activated by cardiolipin in KCs is still unclear. MATERIALS AND METHODS: To make clear of the specific mechanism mentioned above, we firstly used a MCD-induced NASH mice model to demonstrate that CLS1 suppression reduced hepatic steatosis and inflammation. Secondly, the results of IHC staining indicated that the expressions of CLS1 and NLRP3 in liver tissues were significantly upregulated in the NASH group compared to the ND group. On the contrary, CLS1 inhibition significantly downregulated NLRP3 expression in liver tissues, which indicated that CLS1 probably regulated the level of NLRP3 expression. Furthermore, we demonstrated that CLS1 suppression significantly ameliorated the liver function and decreased the TG level, and interleukin-1ß (IL-1ß) and IL-18 were markedly reduced upon CLS1 inhibition. RESULTS: In this work, we reported that cardiolipin is involved in the development of NASH, and the suppression of the cardiolipin synthesis by shRNA-CLS1 could ameliorate the hepatic pathogenic manifestations, as well as the serum inflammatory biomarkers. We further showed that the protein expressions of CLS1, NLRP3, ASC, and Caspase-1 were all upregulated in the NASH liver tissues and palmitic stimulated KCs. CONCLUSIONS: Our study showed that the upregulation of NLRP3 inflammasome activated by cardiolipin is crucial in NASH pathogenesis, which might provide a novel potential role of cardiolipin blockade in the treatment of NASH.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cardiolipinas/metabolismo , Caspase 1/metabolismo , Inflamassomos/metabolismo , Macrófagos do Fígado/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Inflamação , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , RNA Interferente Pequeno , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Triglicerídeos/metabolismo
12.
Med Sci Monit ; 25: 7634-7644, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31603145

RESUMO

BACKGROUND Lysosomal storage diseases (LSDs), a group of rare inherited metabolic disorders, result from specific lysosomal proteins deficiencies in the degradation of biomacromolecule, including over 70 different diseases, most of which are autosomal recessive. LSDs are multisystem disorders, and the clinical manifestations are usually broad and severe, involving the skeletal system, central nervous system (CNS), cardiovascular system, etc. Besides, patients with some subtypes of LSD have distinctive facial features. MATERIAL AND METHODS We performed next generation sequencing on 4 suspected mucopolysaccharidosis (MPS) cases to determine the genetic causes of the disease. By in vitro molecular cell assay, such as real-time polymerase chain reaction (RT-PCR) and western blot, we tested the pathogenicity of candidate variants. RESULTS We detected 5 novel mutations in 4 patients. The mutations were: c.211_214del and c.1270C>T in GUSB; c.1284+1C>A and c.2404C>T in GNPTAB; and c.717C>A in FUCA1). We identified a rare mucopolysaccharidosis VII patient, a rare fucosidosis patient, and 2 rare mucolipidosis II patients, one of which was an atypical patient. We also present a new pathogenic conjecture about a small deletion in GUSB. CONCLUSIONS Our study described rare diseases in Chinese patients and our results enrich the phenotype spectrum of related diseases, as well as mutation spectrum of related genes, which might be significant for clinical disease diagnosis and prenatal diagnosis.


Assuntos
Predisposição Genética para Doença , Doenças por Armazenamento dos Lisossomos/genética , Mutação/genética , Doenças Raras/genética , Sequência de Bases , Criança , Pré-Escolar , Evolução Fatal , Feminino , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Fenótipo , Transferases (Outros Grupos de Fosfato Substituídos)/genética
13.
Biomed Res ; 40(5): 189-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31597904

RESUMO

Sphingomyelin is a major lipid of the plasma membrane and is enriched in microdomains of the plasma membrane that are critical for signal transduction. However, the function of sphingomyelin in the cell membrane of osteoblasts has not been clarified. Therefore, we examined how sphingomyelin synthase 2 (SMS2) affects osteoclast differentiation by osteoblasts. We knocked down the expression of SMS2 with siRNA targeting the Sgms2 gene in mouse primary osteoblasts. The effects of SMS2 knockdown in osteoblasts were examined using polymerase chain reaction and western blotting. The knockdown of SMS2 suppressed the formation of TRAP-positive multinucleated cells by co-culture of osteoblasts and bone marrow cells compared to the control. We found that receptor activator of nuclear factor κB ligand (RANKL) mRNA expression was significantly reduced by 1,25(OH)2D3 stimulation in SMS2 siRNA osteoblasts. The knockdown of SMS2 repressed the expression of retinoid-X-receptor-α (RXRα) regardless of 1,25(OH)2D3 stimulation. TRAP-positive multinucleated cell formation was significantly reduced by RXRα siRNA in osteoblasts in a co-culture system. These results suggest that SMS2 regulates osteoclast differentiation by inducing RANKL expression via RXRα.


Assuntos
Regulação da Expressão Gênica , Osteoblastos/metabolismo , Osteogênese/genética , Ligante RANK/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Inativação Gênica , Camundongos , Osteoclastos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo
14.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548316

RESUMO

Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.


Assuntos
Vacinas Bacterianas/imunologia , Infecções por Erysipelothrix/prevenção & controle , Erysipelothrix/genética , Erysipelothrix/imunologia , Doenças dos Suínos/prevenção & controle , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Elementos de DNA Transponíveis/genética , Erysipelothrix/patogenicidade , Infecções por Erysipelothrix/imunologia , Feminino , Camundongos , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Vacinas Atenuadas/imunologia
15.
J Ind Microbiol Biotechnol ; 46(12): 1745-1755, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31471782

RESUMO

Cell surface engineering was proven as the efficient strategy for enhanced production of target metabolites. In this study, we want to improve the yield of target protein by engineering cell surface in Bacillus licheniformis. First, our results confirmed that deletions of D-alanyl-lipoteichoic acid synthetase gene dltD, cardiolipin synthase gene clsA and CDP-diacylglycerol-serine O-phosphatidyltransferase gene pssA were not conducive to cell growth, and the biomass of gene deletion strains were, respectively, decreased by 10.54 ± 1.43%, 14.17 ± 1.51%, and 17.55 ± 1.28%, while the concentrations of total extracellular proteins were improved, due to the increases of cell surface net negative charge and cell membrane permeability. In addition, the activities of target proteins, nattokinase, and α-amylase were also improved significantly in gene deletion strains. Furthermore, the triplicate gene (dltD, clsA, and pssA) deletion strain was constructed, which further led to the 45.71 ± 2.43% increase of cell surface net negative charge and 26.45 ± 2.31% increase of cell membrane permeability, and the activities of nattokinase and α-amylase reached 37.15 ± 0.89 FU/mL and 305.3 ± 8.4 U/mL, increased by 46.09 ± 3.51% and 96.34 ± 7.24%, respectively. Taken together, our results confirmed that cell surface engineering via deleting dltD, clsA, and pssA is an efficient strategy for enhanced production of target proteins, and this research provided a promising host strain of B. licheniformis for efficient protein expression.


Assuntos
Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Membrana Celular/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Engenharia de Proteínas , Subtilisinas/genética , Subtilisinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo
16.
Mol Biotechnol ; 61(11): 836-851, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31482467

RESUMO

Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693 bp encoding a 230-amino acid protein with a molecular weight of 25.3 kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus. The sfp gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30 °C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn2+, Cu2+, and Fe2+ ions improved the enzymatic activity, while other metals such as Ni2+, Co2+, and Mg2+ had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52 mg/mL, 35.33 U/mg, 3.64 s-1, and 0.104 mM-1 s-1 for Km, Vmax, kcat, and kcat/Km, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5 days of incubation at 37 °C. The recombinant Escherichia coli strain also exhibited an improvement in biosurfactant yield (1.11 g/L) when contrasted with 0.52 g/L from Paenibacillus sp. D9. High esterase activity of 2.55 IU/mL using p-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.


Assuntos
Proteínas de Bactérias/metabolismo , Lipopeptídeos/biossíntese , Paenibacillus/enzimologia , Tensoativos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Esterases/metabolismo , Cinética , Metais Pesados/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Tensoativos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/isolamento & purificação
17.
Nat Commun ; 10(1): 3938, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477732

RESUMO

The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm's pharynx, a bacterial suspension is sucked into the pharynx's lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.


Assuntos
Caenorhabditis elegans/metabolismo , Colesterol/metabolismo , Faringe/metabolismo , Esfingomielinas/metabolismo , Animais , Bactérias/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Mutação , Faringe/citologia , Esfingomielinas/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(35): 17515-17524, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31405983

RESUMO

Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and ß (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.


Assuntos
Astrócitos/metabolismo , Corpo Caloso/metabolismo , Mutação , Gagueira/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Vocalização Animal , Animais , Contagem de Células , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Diester Fosfórico Hidrolases/sangue
19.
J Bacteriol ; 201(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31427391

RESUMO

Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella.IMPORTANCE H. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.


Assuntos
Flagelos/genética , Helicobacter pylori/genética , Proteínas de Membrana/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Alelos , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Mutagênese/genética , Mutação/genética , Transcriptoma/genética
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