RESUMEN
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder. Half of the patients would slowly progress to end-stage renal disease. However, the potential target for ADPKD treatment is still lacking. METHODS: Four ADPKD patients and two healthy family members were included in this study. The peripheral blood samples were obtained and tested by the whole exome sequencing (WES). The autosomal mutations in ADPKD patients were retained as candidate sites. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction network (PPI) analyses were performed by clusterProfiler R package. A dataset containing 18 ADPKD patients and three normal samples were downloaded from the Gene Expression Omnibus (GEO) database and analyzed using the limma R package. RESULTS: A total of six mutant genes were identified based on the dominant genetic pattern and most of them had not been reported to be associated with ADPKD. Furthermore, 19 harmful genes were selected according to the harmfulness of mutation. GO and KEGG enrichment analyses showed that the processes of single-organism cellular process, response to stimulus, plasma membrane, cell periphery, and anion binding as well as cyclic adenosine monophosphate (cAMP) signaling pathway and pathways in cancer were significantly enriched. Through integrating PPI and gene expression analyses, acyl-CoA thioesterase 13 (ACOT13), which has not been reported to be related to ADPKD, and prostaglandin E receptor 2 (PTGER2) were identified as potential genes associated with ADPKD. CONCLUSIONS: Through combination of WES, gene expression, and PPI network analyses, we identified ACOT13 and PTGER2 as potential ADPKD-related genes.
Asunto(s)
Secuenciación del Exoma/métodos , Regulación de la Expresión Génica , Riñón Poliquístico Autosómico Dominante/genética , ARN/genética , Subtipo EP2 de Receptores de Prostaglandina E/genética , Tioléster Hidrolasas/genética , Femenino , Humanos , Masculino , Linaje , Riñón Poliquístico Autosómico Dominante/diagnóstico , Riñón Poliquístico Autosómico Dominante/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/biosíntesis , Tioléster Hidrolasas/biosíntesis , Tomografía Computarizada por Rayos XRESUMEN
Sertoli cells are a type of nurse cell in the seminiferous epithelium that are crucial for sustaining spermatogenesis by extending nutritional and energy support to the developing germ cells. Dysfunction of Sertoli cells could cause disordered spermatogenesis and reduced fertility in males. In this study, we focused on the expression and function of palmitoyl protein thioesterase 1 (PPT1), a lysosomal depalmitoylating enzyme, in Sertoli cells. Here, we show that PPT1 expression in Sertoli cells is responsive to cholesterol treatment and that specific knockout of Ppt1 in Sertoli cells causes male subfertility associated with poor sperm quality and a high ratio of sperm deformity. Specifically, Ppt1 deficiency leads to poor cell variably accompanied with abnormal lysosome accumulation and increased cholesterol levels in Sertoli cells. Further, Ppt1 deficiency results in poor adhesion of developing germ cells to Sertoli cells in the seminiferous epithelium, which is likely to be responsible for the reduced male fertility as a consequence of declines in sperm count and motility as well as a high incidence of sperm head deformity. In summary, PPT1 affects sperm quality and male fertility through regulating lysosomal function and cholesterol metabolism in Sertoli cells.
Asunto(s)
Colesterol/metabolismo , Fertilidad , Regulación Enzimológica de la Expresión Génica , Células de Sertoli/enzimología , Espermatozoides/enzimología , Tioléster Hidrolasas/biosíntesis , Animales , Masculino , Ratones , Túbulos Seminíferos/citología , Túbulos Seminíferos/enzimología , Células de Sertoli/citología , Recuento de Espermatozoides , Espermatozoides/citologíaRESUMEN
Abiotic stresses enhance cellular reactive oxygen species (ROS) level which results in toxic methylglyoxal (MG) production. Glyoxalases catalyze the conversion of toxic MG into non-toxic lactic acid whose properties and function are still unknown in cyanobacteria. This is the first attempt to characterize All0580 from Anabaena sp. PCC7120 as GlyII using in silico and wet lab approaches. Data of functional complementation of E. coli GlyII mutant (ΔgloB), enzyme kinetics and ESI-MS analysis suggested that All0580 harbors distinctive GlyII activity. The catalytic efficiency of All0580 (3â¯×â¯106â¯M-1â¯s-1) is higher than Arabidopsis GlyII. AAS analysis revealed the presence of a binuclear Zn/Fe centre in All0580 active site. The qRT-PCR of the target gene revealed maximum up-regulation in salinity followed by drought, arsenic, heat, and UV-B stresses. BL21/pET-21a-all0580 showed 1.5 to 10 fold increased growth and up to 4 fold decreased intracellular MG level as compared to BL21/pET-21a cells under various abiotic stresses and MG. A 39% drop in ROS generation by BL21/pET-21a-all0580 under MG stress suggested its potential to manage MG toxicity. Above attributes suggest that the hypothetical protein All0580 is a novel GlyII of cyanobacteria which heterologously confers tolerance to multiple abiotic stresses in E. coli.
Asunto(s)
Anabaena , Proteínas Bacterianas , Escherichia coli , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Tioléster Hidrolasas , Anabaena/enzimología , Anabaena/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Tioléster Hidrolasas/biosíntesis , Tioléster Hidrolasas/genéticaRESUMEN
OBJECTIVE: We have identified a gene YOD1 encoding deubiquitinating enzyme (DUB) responsible for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We aimed to determine the effects of YOD1 RNA interference (RNAi) on cell proliferation and migration, playing an important role in lip and palate formation, and to clarify whether the mechanisms involved TGF-ß3 signaling associated with NSCL/P. METHODS: RNAi was applied to construct vectors expressing YOD1 small interference RNAs (siRNAs). The vectors were transfected into the human oral keratinocytes (HOK) cells. The cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The mRNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels were investigated by western blotting. RESULTS: The proliferation of YOD1 siRNA-transfected HOK cells was remarkably inhibited. The migration rate was significantly decreased in the YOD1 siRNA-transfected HOK cells. The TGF-ß3 mRNA and protein levels were decreased significantly by siRNA-mediated knockdown of YOD1. YOD1 RNAi reduced the phosphor-Smad2/3 levels significantly. CONCLUSIONS: YOD1 RNAi may inhibit cell proliferation and migration associated with the pathogenesis of NSCL/P through TGF-ß3 signaling. The study indicates a novel role of YOD1 in regulating TGF-ß3 signaling to affect cell proliferation and migration resulting in NSCL/P.
Asunto(s)
Movimiento Celular , Proliferación Celular , Endopeptidasas/biosíntesis , Queratinocitos/metabolismo , Boca/metabolismo , Interferencia de ARN , Transducción de Señal , Tioléster Hidrolasas/biosíntesis , Factor de Crecimiento Transformador beta3/metabolismo , Labio Leporino/genética , Labio Leporino/metabolismo , Labio Leporino/patología , Fisura del Paladar/genética , Fisura del Paladar/metabolismo , Fisura del Paladar/patología , Endopeptidasas/genética , Humanos , Queratinocitos/patología , Boca/patología , Tioléster Hidrolasas/genética , Factor de Crecimiento Transformador beta3/genéticaRESUMEN
USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in chemoresistant HCC tissues and cells and was correlated with the prognosis of HCC patients who received chemotherapy. Silencing USP22 in chemoresistant HCC Bel/Fu cells dramatically inhibited proliferation, migration, invasion and epithelial-mesenchymal transition in vitro; suppressed tumorigenic and metastatic capacities in vivo; and inhibited drug resistance-related proteins (MDR1, LRP, MRP1). Mechanistically, we found that USP22 knockdown exerts its function through down-regulating PI3K and activating Smad4, which inhibited phosphorylation of Akt. Silencing Smad4 blocked USP22 knockdown-induced Akt inhibition in Bel/Fu cells. Our results, for the first time, provide evidence that USP22 plays a critical role in the development of chemoresistant HCC cells and that high USP22 expression serves as a molecular marker for the prognosis of HCC patients who undergo chemotherapy.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Fluorouracilo/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteína Smad4/metabolismo , Tioléster Hidrolasas/deficiencia , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Fluorouracilo/farmacología , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Smad4/genética , Tioléster Hidrolasas/biosíntesis , Tioléster Hidrolasas/metabolismo , Ubiquitina Tiolesterasa , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ubiquitin-specific protease 22 (USP22) aberrance has been implicated in several malignancies; however, whether USP22 plays a role in anaplastic thyroid carcinoma (ATC) remains unclear. Here, we report that USP22 expression is highly elevated in ATC tissues, which positively correlated with tumor size, extracapsular invasion, clinical stages, and poor prognosis of ATC patients. In vitro assays showed that USP22 depletion suppressed ATC cell survival and proliferation by decreasing Rb phosphorylation and cyclin D2, inactivating Akt, and simultaneously upregulating Rb; USP22 silencing restrained cell migration and invasion by inhibiting epithelial-mesenchymal transition; USP22 knockdown promoted mitochondrion- mediated and caspase-dependent apoptosis by upregulating Bax and Bid and promoting caspase-3 activation. Consistent with in vitro findings, downregulation of USP22 in ATC cells impeded tumor growth and lung metastasis in vivo. These results raise the applicability for USP22 as a useful predictor of ATC prognosis and a potential therapeutic target for ATC.
Asunto(s)
Tioléster Hidrolasas/biosíntesis , Carcinoma Anaplásico de Tiroides/enzimología , Neoplasias de la Tiroides/enzimología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Ratones , Ratones SCID , Metástasis de la Neoplasia , Pronóstico , Transducción de Señal , Tioléster Hidrolasas/genética , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Ubiquitina TiolesterasaRESUMEN
Ubiquitin-specific protease 42 (USP42) is a member of deubiquitinating enzymes (DUBs). The alterations of DUBs are implicated in the pathogenesis of a wide variety of tumors. However, there are few studies on the expression and biological function of USP42 in gastric cancer (GC). Here, the expression levels of USP42 were significantly higher in GC tissues than in non-tumorous tissues. USP42 expression was significantly correlated with tumor size, TNM stage, lymph node metastasis and overall survival of patients with GC. Moreover, USP42 silencing in two GC cell lines, AGS and MKN-45, notably inhibited cell proliferation, but stimulated G1 phase arrest. The proteins promoting cell cycle progression (Cyclin D1, Cyclin E1 and PCNA) were down-regulated in USP42-suppressed cells. Moreover, inhibition of USP42 in GC cells impaired cell invasion via affecting the expression of matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition (EMT) regulators. In conclusion, USP42 overexpression could be a potential prognostic marker for GC, regulate the survival and invasive properties of GC, and may represent a novel therapeutic molecular target for this tumor.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Transición Epitelial-Mesenquimal , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/mortalidad , Tioléster Hidrolasas/biosíntesis , Adulto , Anciano , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Neoplasias Gástricas/patología , Tasa de SupervivenciaRESUMEN
Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Esterasas/metabolismo , Pirroles/química , Streptomyces/enzimología , Compuestos de Sulfhidrilo/química , Tioléster Hidrolasas/biosíntesis , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Dominio Catalítico , Clonación Molecular , Biología Computacional , Esterasas/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Filogenia , Streptomyces/genética , Tioléster Hidrolasas/químicaRESUMEN
The conversion of industrial by-products into high-value added compounds is a challenging issue. Crude glycerol, a by-product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol-based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate â¼5 g L(-1) of biomass and 0.8 g L(-1) of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L(-1) of urea or ammonium sulfate and 20 g L(-1) of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium-chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids.
Asunto(s)
Ingeniería Metabólica , Ácido Oléico/biosíntesis , Proteínas de Plantas/biosíntesis , Tioléster Hidrolasas/biosíntesis , Sulfato de Amonio/química , Biomasa , Reactores Biológicos , Carbono/metabolismo , Medios de Cultivo , Cuphea/enzimología , Glicerol/metabolismo , Ácido Oléico/metabolismo , Proteínas de Plantas/metabolismo , Tioléster Hidrolasas/metabolismo , Yarrowia/enzimología , Yarrowia/genéticaRESUMEN
Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor type, ranking as the third leading cause of all cancer-related deaths in the world. The post-surgical 5-year survival rate is low, largely due to the high recurrence rate. Therefore, the identification of target molecules that control the biological characteristics of HCC is of great importance. Ubiquitin-specific protease 22 (USP22) is a newly discovered deubiquitinating enzyme and is a cancer stem cell marker that plays a role in tumorigenesis, therapy resistance and cell cycle progression. Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is known to function either as an inhibitor for apoptosis or as a regulator of cell division. Levels of survivin are correlated with the aggressiveness of tumors and a poor prognosis in various cancers including HCC. In the present study, we examined the USP22 expression and its association with survivin expression and clinicopathological features in HCC. First, we examined the expression of USP22 and survivin in 151 HCC cases by immunohistochemistry. High expression of USP22 and survivin was frequently observed in HCC cases, in comparison with normal adjacent liver tissues. Expression of USP22 and survivin was well correlated with malignant behavior including tumor size, stage and differentiation in HCC cases. Importantly, HCC patients with high expression of USP22 and survivin showed poor prognosis. USP22 expression was well correlated with survivin expression in HCC cases. This correlation was confirmed in HCC cell lines and tissues by RT-PCR and western blot analysis. Next, to investigate the biological role of USP22 in HCC, we examined the effect of USP22 knockdown on the cell growth and the expression of cell cycle-related protein including survivin in HCC cells. USP22 siRNA suppressed cell growth. Moreover, USP22 siRNA decreased survivin expression together with upregulation of CDK inhibitor, p21 and downregulation of cyclin B. These findings suggest that USP22 may be involved in HCC progression in cooperation with survivin. We suggest that USP22 can be useful as a new prognostic marker and therapeutic target in HCC patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/patología , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Neoplasias Hepáticas/patología , Tioléster Hidrolasas/biosíntesis , Adulto , Anciano , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/análisis , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Tioléster Hidrolasas/análisis , Transfección , Ubiquitina Tiolesterasa , Adulto JovenRESUMEN
Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species.
Asunto(s)
Citrus , Lactoilglutatión Liasa , Proteínas de Plantas , Raíces de Plantas , Plantas Modificadas Genéticamente , Tolerancia a la Sal , Tioléster Hidrolasas , Citrus/enzimología , Citrus/genética , Lactoilglutatión Liasa/biosíntesis , Lactoilglutatión Liasa/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Tioléster Hidrolasas/biosíntesis , Tioléster Hidrolasas/genéticaRESUMEN
In this study, we examined the possible mechanisms of trehalose (Tre) in improving copper-stress (Cu-stress) tolerance in rice seedlings. Our findings indicated that pretreatment of rice seedlings with Tre enhanced the endogenous Tre level and significantly mitigated the toxic effects of excessive Cu on photosynthesis- and plant growth-related parameters. The improved tolerance induced by Tre could be attributed to its ability to reduce Cu uptake and decrease Cu-induced oxidative damage by lowering the accumulation of reactive oxygen species (ROS) and malondialdehyde in Cu-stressed plants. Tre counteracted the Cu-induced increase in proline and glutathione content, but significantly improved ascorbic acid content and redox status. The activities of major antioxidant enzymes were largely stimulated by Tre pretreatment in rice plants exposed to excessive Cu. Additionally, increased activities of glyoxalases I and II correlated with reduced levels of methylglyoxal in Tre-pretreated Cu-stressed rice plants. These results indicate that modifying the endogenous Tre content by Tre pretreatment improved Cu tolerance in rice plants by inhibiting Cu uptake and regulating the antioxidant and glyoxalase systems, and thereby demonstrated the important role of Tre in mitigating heavy metal toxicity. Our findings provide a solid foundation for developing metal toxicity-tolerant crops by genetic engineering of Tre biosynthesis.
Asunto(s)
Antioxidantes/farmacología , Cobre/toxicidad , Oryza/efectos de los fármacos , Plantones/efectos de los fármacos , Trehalosa/farmacología , Ácido Ascórbico/agonistas , Ácido Ascórbico/biosíntesis , Transporte Biológico/efectos de los fármacos , Glutatión/agonistas , Glutatión/biosíntesis , Lactoilglutatión Liasa/biosíntesis , Malondialdehído/metabolismo , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Fotosíntesis/efectos de los fármacos , Prolina/agonistas , Prolina/biosíntesis , Piruvaldehído/antagonistas & inhibidores , Piruvaldehído/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Estrés Fisiológico , Tioléster Hidrolasas/biosíntesisRESUMEN
Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoR I/Xba I, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZaA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.
Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Arabidopsis/genética , Tioléster Hidrolasas/biosíntesis , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Electroporación , Pichia/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Transformación GenéticaRESUMEN
Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.
Asunto(s)
Proteínas Bacterianas , Caprilatos/farmacología , Evolución Molecular Dirigida , Farmacorresistencia Bacteriana , Firmicutes/genética , Tioléster Hidrolasas , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Firmicutes/metabolismo , Concentración de Iones de Hidrógeno , Tioléster Hidrolasas/biosíntesis , Tioléster Hidrolasas/genéticaRESUMEN
Ubiquitin-specific protease 22 (USP22) removes ubiquitin from histones, thus regulating gene transcription. The expression frequency and expression levels of USP22 were significantly higher in hepatocellular carcinoma (HCC) than in normal liver tissues. High USP22 expression in HCC was significantly correlated with clinical stage and tumor grade. Kaplan-Meier analysis showed that elevated USP22 expression predicted poorer overall survival and recurrence-free survival. High USP22 expression was also associated with shortened survival time in patients at advanced tumor stages and with high grade HCC. Multivariate analyses revealed that USP22 expression is an independent prognostic parameter in HCC. These findings provide evidence that high USP22 expression might be important in tumor progression and serves as an independent molecular marker for poor HCC prognosis. Thus, USP22 overexpression identifies patients at high risk and represents a novel therapeutic molecular target for this tumor.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Tioléster Hidrolasas/biosíntesis , Adulto , Western Blotting , Carcinoma Hepatocelular/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pronóstico , Tioléster Hidrolasas/análisis , Ubiquitina TiolesterasaRESUMEN
Ubiquitinspecific protease 22 (USP22) is a component of the transcription regulatory histone acetylation complex SAGA, which broadly regulates gene transcription and correlates with cancer progression, metastasis and prognosis. Autophagy is a cell pathway with dual functions that promotes cell survival or death. However, it is not known whether USP22 can regulate autophagy in pancreatic cancer. In the present study, we first identified that USP22 was overexpressed in a large number of pancreatic cancer patient samples, concomitant with the increased expression of LC3, a marker of autophagy. Statistical analysis revealed that the increase in USP22 and autophagy was positively correlated with poor prognosis of pancreatic cancer patients. Further investigation using a human pancreatic cancer cell (Panc1) identified that the overexpression of USP22 increased the processing of LC3 into the active form LC3II and the number of autophagosomes, thus leading to enhanced autophagy. Activation of ERK1/2 kinase rather than AKT1 by USP22 was found to be one of the mechanisms promoting LC3 processing. USP22induced autophagy was also found to enhance cell proliferation and resistance to starvation and chemotherapeutic drugs in Panc1 cells, therefore expressing an overall effect that promotes cell survival. Collectively, the present study demonstrated a new function of USP22 that induces autophagy, thus leading to the poor prognosis of pancreatic cancer.
Asunto(s)
Autofagia/genética , Neoplasias Pancreáticas/genética , Pronóstico , Tioléster Hidrolasas/biosíntesis , Anciano , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Especies Reactivas de Oxígeno/metabolismo , Tioléster Hidrolasas/genética , Ubiquitina TiolesterasaRESUMEN
Acyl-acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl-ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61-73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30 a vector and transformed into Escherichia coli BL21(DE3)â³FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~40.5kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl-ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.
Asunto(s)
Lindera/enzimología , Tioléster Hidrolasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Ácidos Decanoicos/metabolismo , Perfilación de la Expresión Génica , Ácidos Láuricos/metabolismo , Datos de Secuencia Molecular , Filogenia , Semillas/enzimología , Alineación de Secuencia , Análisis de Secuencia de ADN , Tioléster Hidrolasas/biosíntesis , Tioléster Hidrolasas/clasificación , Tioléster Hidrolasas/metabolismoRESUMEN
PURPOSE: To detect the expression levels of ubiquitin-specific protease 22 (USP22) in human esophageal squamous cell carcinoma (ESCC) and to correlate it with clinicopathologic and prognostic data. METHODS: The immunoreactivity of USP22 protein was analyzed in 157 pathologically characterized ESCC tissues by immunohistochemistry. All statistical analyses were performed with SPSS statistical software to evaluate the association of USP22 protein with clinicopathologic factors and survival. RESULTS: High expression of USP22 protein was detected in 50.96 % of 157 ESCC tissues and significantly associated with invasion depth, lymph node metastasis, pathologic stage and tumor relapse (P < 0.05, respectively). Univariate survival analysis showed that patients with high expression of USP22 protein had a significantly poorer 5-year disease-specific survival (P = 0.002), and multivariate survival analysis showed that high expression of USP22 protein was an independent prognosticator for unfavorable disease-specific survival (P = 0.039). Further survival analysis stratified by pathologic stage demonstrated that high expression of USP22 protein significantly predicted unfavorable clinical outcome (P = 0.029) among patients with pathologic stage II(b)-III diseases. CONCLUSION: USP22 protein plays an essential role in ESCC progression and has clinical potentials not only as a promising biomarker to identify the subgroup of patients with more aggressive tumors and poor prognostic potential but also as an attractively therapeutic target for ESCC.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Núcleo Celular/enzimología , Neoplasias Esofágicas/enzimología , Tioléster Hidrolasas/biosíntesis , Biomarcadores de Tumor/biosíntesis , Carcinoma de Células Escamosas/patología , Progresión de la Enfermedad , Neoplasias Esofágicas/patología , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Ubiquitina TiolesterasaRESUMEN
OBJECTIVE: Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited childhood neurodegenerative disorder caused by the loss of palmitoyl protein thioesterase-1 (PPT1) activity. Affected children suffer from blindness, epilepsy, motor dysfunction, cognitive decline, and premature death. The Ppt1(-/-) mouse shares the histological and clinical features of INCL. Previous single-therapy approaches using small molecule drugs, gene therapy, or neuronal stem cells resulted in partial histological correction, with minimal improvements in motor function or lifespan. Here, we combined central nervous system (CNS)-directed adeno-associated virus (AAV)2/5-mediated gene therapy with bone marrow transplantation (BMT) in the INCL mouse. METHODS: At birth, Ppt1(-/-) and wild-type mice were given either intracranial injections of AAV2/5-PPT1 or bone marrow transplantation, separately as well as in combination. To assess function, we measured rotorod performance monthly as well as lifespan. At terminal time points, we evaluated the therapeutic effects on several INCL-specific parameters, such as cortical thickness, autofluorescent accumulation, and glial activation. Finally, we determined levels of PPT1 enzyme activity and bone marrow engraftment in treated mice. RESULTS: AAV2/5-mediated gene therapy alone resulted in significant histological correction, improved motor function, and increased lifespan. Interestingly, the addition of BMT further increased the lifespan of treated mice and led to dramatic, sustained improvements in motor function. These data are truly striking, given that BMT alone is ineffective, yet it synergizes with CNS-directed gene therapy to dramatically increase efficacy and lifespan. INTERPRETATION: AAV2/5-mediated gene therapy in combination with BMT provides an unprecedented increase in lifespan as well as dramatic improvement on functional and histological parameters.
Asunto(s)
Trasplante de Médula Ósea/métodos , Terapia Genética/métodos , Lipofuscinosis Ceroideas Neuronales/terapia , Tioléster Hidrolasas/biosíntesis , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Encéfalo/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Lipofuscinosis Ceroideas Neuronales/patología , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Prueba de Desempeño de Rotación con Aceleración Constante , Tioléster Hidrolasas/deficiencia , Tioléster Hidrolasas/uso terapéuticoRESUMEN
HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS.