RESUMO
Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O2 evolution and CO2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.
Assuntos
Glicogênio , Fotossíntese , Complexo de Proteína do Fotossistema II , Synechocystis , Synechocystis/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/crescimento & desenvolvimento , Synechocystis/genética , Glicogênio/metabolismo , Transporte de Elétrons , Complexo de Proteína do Fotossistema II/metabolismo , Mutação/genética , Glucose/metabolismo , Dióxido de Carbono/metabolismo , Oxigênio/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Fosfoglucomutase/metabolismo , Fosfoglucomutase/genéticaRESUMO
Pneumocystis cyst life forms contain abundant ß-glucan carbohydrates, synthesized using ß-1,3 and ß-1,6 glucan synthase enzymes and the donor uridine diphosphate (UDP)-glucose. In yeast, phosphoglucomutase (PGM) plays a crucial role in carbohydrate metabolism by interconverting glucose 1-phosphate and glucose 6-phosphate, a vital step in UDP pools for ß-glucan cell wall formation. This pathway has not yet been defined in Pneumocystis. Herein, we surveyed the Pneumocystis jirovecii and Pneumocystis murina genomes, which predicted a homolog of the Saccharomyces cerevisiae major PGM enzyme. Furthermore, we show that PjPgm2p and PmPgm2p function similarly to the yeast counterpart. When both Pneumocystis pgm2 homologs are heterologously expressed in S. cerevisiae pgm2Δ cells, both genes can restore growth and sedimentation rates to wild-type levels. Additionally, we demonstrate that yeast pgm2Δ cell lysates expressing the two Pneumocystis pgm2 transcripts individually can restore PGM activities significantly altered in the yeast pgm2Δ strain. The addition of lithium, a competitive inhibitor of yeast PGM activity, significantly reduces PGM activity. Next, we tested the effects of lithium on P. murina viability ex vivo and found the compound displays significant anti-Pneumocystis activity. Finally, we demonstrate that a para-aryl derivative (ISFP10) with known inhibitory activity against the Aspergillus fumigatus PGM protein and exhibiting 50-fold selectivity over the human PGM enzyme homolog can also significantly reduce Pmpgm2 activity in vitro. Collectively, our data genetically and functionally validate phosphoglucomutases in both P. jirovecii and P. murina and suggest the potential of this protein as a selective therapeutic target for individuals with Pneumocystis pneumonia.
Assuntos
Pneumocystis carinii , Pneumocystis , Pneumonia por Pneumocystis , beta-Glucanas , Humanos , Pneumocystis carinii/genética , Pneumonia por Pneumocystis/tratamento farmacológico , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Fosfoglucomutase/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Lítio/metabolismo , Lítio/farmacologia , Pneumocystis/genética , beta-Glucanas/metabolismo , Fosfatos/farmacologia , Glucose/metabolismo , Difosfato de Uridina/metabolismo , Difosfato de Uridina/farmacologiaRESUMO
Spondylo-epi-metaphyseal dysplasias (SEMDs) are a clinically and genetically heterogeneous group of skeletal dysplasias characterized by short stature and abnormal modeling of the spine and long bones. A novel form of rhizomelic skeletal dysplasia, Ain-Naz type, associated with a homozygous variant in GNPNAT1 was recently identified. Herein, we report an Egyptian patient, offspring of consanguineous parents, who presented with a severe form of unclassified SEMD. Whole exome sequencing identified a novel homozygous variant in exon 3, c.77T>G, (p.Phe26Cys) in GNPNAT1, that was confirmed by Sanger sequencing and both parents were found to be heterozygous for the identified variant. Main features included severe short stature, rhizomelic limb shortening, and wide flared metaphysis. Short broad long bones, brachydactyly, delayed epiphyseal ossification of long bones, advanced bone age, and immunodeficiency were additional findings expanding the clinical phenotype described in the previously reported family. We conclude that variants in the GNPNAT1 gene cause an autosomal recessive form of SEMD resembling Desbuquois like dysplasia caused by PGM3, which is involved in the same pathway as GNPNAT1.
Assuntos
Nanismo , Osteocondrodisplasias , Nanismo/diagnóstico por imagem , Nanismo/genética , Glucosamina 6-Fosfato N-Acetiltransferase/genética , Heterozigoto , Humanos , Hiperplasia , Osteocondrodisplasias/genética , Fosfoglucomutase/genética , Sequenciamento do ExomaRESUMO
OBJECTIVE: Toxoplasma gondii is a ubiquitous parasite of medical and veterinary importance; however, there exists no cure for chronic toxoplasmosis. Metabolic enzymes required for the production and maintenance of tissue cysts represent promising targets for novel therapies. Here, we use reverse genetics to investigate the role of Toxoplasma phosphoglucomutase 1, PGM1, in Toxoplasma growth and cystogenesis. RESULTS: We found that disruption of pgm1 did not significantly affect Toxoplasma intracellular growth and the lytic cycle. pgm1-defective parasites could differentiate into bradyzoites and produced cysts containing amylopectin in vitro. However, cysts produced in the absence of pgm1 were significantly smaller than wildtype. Together, our findings suggest that PGM1 is dispensable for in vitro growth but contributes to optimal Toxoplasma cyst development in vitro, thereby necessitating further investigation into the function of this enzyme in Toxoplasma persistence in its host.
Assuntos
Fosfoglucomutase , Toxoplasma , Toxoplasmose , Humanos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Toxoplasma/enzimologia , Toxoplasma/genética , Toxoplasmose/parasitologiaRESUMO
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 µM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Glucanos/metabolismo , Humanos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismoRESUMO
In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and tumor suppressor STK11 (also known as LKB1) confer an aggressive malignant phenotype, an unfavourability towards immunotherapy, and overall poor prognoses in patients. In a previous study, we showed that murine KRAS/LKB1 co-mutant tumors and human co-mutant cancer cells have an enhanced dependence on glutamine-fructose-6-phosphate transaminase 2 (GFPT2), a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP), which could be targeted to reduce survival of KRAS/LKB1 co-mutants. Here, we found that KRAS/LKB1 co-mutant cells also exhibit an increased dependence on N-acetylglucosamine-phosphate mutase 3 (PGM3), an enzyme downstream of GFPT2. Genetic or pharmacologic suppression of PGM3 reduced KRAS/LKB1 co-mutant tumor growth in both in vitro and in vivo settings. Our results define an additional metabolic vulnerability in KRAS/LKB1 co-mutant tumors to the HBP and provide a rationale for targeting PGM3 in this aggressive subtype of NSCLC.
Assuntos
Quinases Proteína-Quinases Ativadas por AMP/genética , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Fosfoglucomutase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glicosilação/efeitos dos fármacos , Hexosaminas/biossíntese , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Fosfoglucomutase/antagonistas & inibidores , Fosfoglucomutase/genéticaRESUMO
Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Complexos Multienzimáticos/química , Fosfoglucomutase/química , Fósforo-Oxigênio Liases/química , Multimerização Proteica , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Complexos Multienzimáticos/genética , Fosfoglucomutase/genética , Fósforo-Oxigênio Liases/genética , Domínios Proteicos , Estrutura Quaternária de ProteínaRESUMO
Phosphoglucomutase 3 (PGM3) deficiency is a rare congenital disorder of glycosylation. Most of patients with autosomal recessive hypomorphic mutations in PGM3 encoding for phosphoglucomutase 3 present with eczema, skin and lung infections, elevated serum IgE, as well as neurological and skeletal features. A few PGM3-deficient patients suffer from a more severe disease with nearly absent T cells and severe skeletal dysplasia. We performed targeted next-generation sequencing on two kindred to identify the underlying genetic etiology of a severe combined immunodeficiency with developmental defect. We report here two novel homozygous missense variants (p.Gly359Asp and p.Met423Thr) in PGM3 identified in three patients from two unrelated kindreds with severe combined immunodeficiency, neurological impairment, and skeletal dysplasia. Both variants segregated with the disease in the two families. They were predicted to be deleterious by in silico analysis. PGM3 enzymatic activity was found to be severely impaired in primary fibroblasts and Epstein-Barr virus immortalized B cells from the kindred carrying the p.Met423Thr variant. Our findings support the pathogenicity of these two novel variants in severe PGM3 deficiency.
Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Deformidades Congênitas dos Membros/genética , Doenças do Sistema Nervoso/genética , Fosfoglucomutase/genética , Imunodeficiência Combinada Severa/genética , Pré-Escolar , Face/anormalidades , Feminino , Humanos , Lactente , Recém-Nascido , MasculinoRESUMO
Background: Bioinformatics showed that long non-coding RNA (lncRNA) pgm5-as1 was regulated in patients with colorectal cancer (CRC), and miR-484 was also regulated in CRC. We aimed at determining the modulatory pathway of lncRNA pgm5-as1 in CRC cells, and whether miR-484 was involved in the pathway. Materials and Methods: The target gene of pgm5-as1 was predicted by bioinformatics and verified by dual luciferase assay. Transcription levels of pgm5-as1 and miR-484 were determined by quantitative real-time polymerase chain reaction. Viability, migration rate, invasion, and growth of SW480 and HCT116 cells were determined by Cell Counting Kit-8 (CCK-8), wound healing assay, transwell, and colony formation assay, respectively. Results: pgm5-as1 was upregulated in CRC tissues and cell lines; however, its downregulation contributed to the decreasing of cell viability, growth, migration, and invasion of SW480 and HCT116 cells. Moreover, miR-484 was predicted as the target of pgm5-as1, and the downregulation of pgm5-as1 partially restored the elevated cell viability, growth, migration, and invasion that were induced by the inhibition of miR-484 expression in SW480 and HCT116 cells. Conclusions: The loss of miR-484 expression in CRC might be involved in the promotion and metastasis of CRC, which may be caused by the overexpression of pgm5-as1. Hence, the downregulation of pgm5-as1 could be a therapeutic target in the prevention or intervention of CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas do Citoesqueleto/genética , MicroRNAs/metabolismo , Fosfoglucomutase/genética , RNA Longo não Codificante/metabolismo , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Metástase Neoplásica , Fosfoglucomutase/metabolismo , RNA Longo não Codificante/genéticaRESUMO
Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.
Assuntos
Adenosina Trifosfatases/genética , Antimaníacos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cloreto de Lítio/farmacologia , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoglucomutase/genética , Biossíntese de Proteínas/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In solid tumors, hypoxia can trigger aberrant expression of transcription factors and genes, resulting in abnormal biological functions such as altered energetic pathways in cancer cells. Glucose metabolism is an important part of this phenomenon, which is associated with changes in the functional expression of transporters and enzymes involved in the glycolysis pathway. The latter phenomenon can finally lead to the lactate accumulation and pH dysregulation in the tumor microenvironment and subsequently further invasion and metastasis of cancer cells. Having capitalized on the computational modeling, in this study, for the first time, we aimed to investigate the effects of hypoxia-induced factor-1 (HIF-1) mediated hypoxia on the magnitude of functional expression of all the enzymes and transporters involved in the glycolysis process. The main objective was to establish a quantitative relationship between the hypoxia intensity and the intracellular lactate levels and determine the key regulators of the glycolysis pathway. This model clearly showed an increase in the lactate concentration during the oxygen depletion. The proposed model also predicted that the phosphofructokinase-1 and phosphoglucomutase enzymes might play the most important roles in the regulation of the lactate production.
Assuntos
Glicólise/genética , Hipóxia/genética , Hipóxia/metabolismo , Ácido Láctico/metabolismo , Modelos Teóricos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/genética , Microambiente Tumoral , Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Neoplasias/patologia , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/fisiologia , Fosfoglucomutase/genética , Fosfoglucomutase/fisiologiaRESUMO
Cancer cells undergo metabolic reprogramming to sustain their own survival under an environment of increased energy demand; however, the mechanism by which cancer cells ensure survival under glucose deprivation stressed conditions remains elusive. Here, we show that deprivation of glucose, dramatically activated the glycogen pathway, accompanied by elevated phosphoglucomutase 1 (PGM1) expression. We further identified that AMP-activated protein kinase (AMPK) stimulated PGM1 expression by inducing histone deacetylase 8 (HDAC8) phosphorylation. Moreover, we demonstrated that glucose deprivation-induced AMPK activation stimulated the translocation of HDAC8 from the nucleus to the cytoplasm, consequently disrupting the binding between HDAC8 and histone 3. PGM1 expression was also found to be critical for lung cancer glycolysis, the oxidative pentose phosphate pathway, and oxidative phosphorylation under glucose deprivation conditions, and further led to the aberrant expression of metabolic enzymes involved in glucose metabolism mediated by ERK1/2. Finally, PGM1 was found to be highly expressed in lung cancer tissues from patients, which correlated with a poor prognosis. Taken together, these results revealed that AMPK activation by glucose deprivation leads to enhanced PGM1 expression, an essential component of the metabolic switch, to facilitate cancer progression, suggesting PGM1 as promising anti-cancer treatment targets.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Histona Desacetilases/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Proteínas Repressoras/metabolismo , Células A549 , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicólise , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Fosforilação , Transdução de Sinais , Regulação para CimaRESUMO
OBJECTIVE: Growing studies have shown that long non-coding RNAs (lncRNAs) have critical regulatory roles in tumorigenesis. Recently, a newly identified lncRNA, Homo sapiens PGM5 antisense RNA 1 (PGM5-AS1), was found to be dysregulated in several tumors. However, its roles in clear cell renal cell carcinoma (ccRCC) have not been investigated. The aim of the present study was to clarify the clinical significance of PGM5-AS1 in ccRCC patients. PATIENTS AND METHODS: The PGM5-AS1 expression levels were evaluated in 182 primary ccRCC patients using quantitative real-time PCR assays. The associations between expression of PGM5-AS1, clinicopathological parameters, and prognosis of ccRCC were examined using Chi-square test, Kaplan-Meier assays, and multivariate assays. RESULTS: The expressions of PGM5-AS1 in cancer specimens were lower than those in matched non-tumor specimens from the ccRCC patient (p<0.05). Downregulation of PGM5-AS1 was closely associated with more advanced clinical features, including lymph nodes metastasis (p=0.007) and distant metastasis (p=0.037). A clinical study revealed that ccRCC patients with lower PGM5-AS1 expressions had substantially shorter overall survival (OS) and disease-free survival (DFS) than patients with higher PGM5-AS1 expressions. Further multivariate assays demonstrated that PGM5-AS1 was identified as an independent prognostic factor for patients with ccRCC. CONCLUSIONS: Down-regulation of PGM5-AS1 in ccRCC tissues had a strong association with unfavorable outcomes and PGM5-AS1 might be a potential tumor suppressor.
Assuntos
Carcinogênese/genética , Carcinoma de Células Renais/genética , Proteínas do Citoesqueleto/genética , Neoplasias Renais/genética , Fosfoglucomutase/genética , RNA Longo não Codificante/genética , Carcinoma de Células Renais/patologia , Estudos de Coortes , Regulação para Baixo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
BACKGROUND: Excessive alcohol consumption is a major public health problem in East Asian countries. Alcohol use leads to a cascade of problems including increased chances of risky behavior and a wide range of negative health consequences, from alcoholic liver disease to upper gastric and liver cancer. These alcohol effects are known to be influenced by ethnic variability and genetics. METHODS: In this study, subjects were administered a single dose of alcohol (0.6 g/kg for men or 0.4 g/kg for women), and blood alcohol and acetaldehyde concentrations were measured eight times over 5 hours. To investigate genetically susceptible factors to alcohol metabolism, we selected single-nucleotide polymorphisms (SNP) of genes identified by prior genetic association studies for alcohol metabolism, alcohol consumption, alcohol dependence, and related traits, and performed genotyping on all subjects (n = 104). RESULTS: We identified variations in the ADH1A, SRPRB, and PGM1 genes, which are directly associated with blood alcohol or acetaldehyde concentrations. Namely, the T allele of SRPRB rs17376019 and the C allele of PGM1 rs4643 were associated with lower blood alcohol levels, while the ADH1 rs1229976 C allele group exhibited markedly higher blood acetaldehyde levels than those of the ADH1 rs1229976 T allele group. CONCLUSION: This study demonstrates that genetic variations in ADH1A, SRPRB, and PGM1 are associated with variations in blood alcohol and acetaldehyde concentration after alcohol intake.
Assuntos
Álcool Desidrogenase/genética , Consumo de Bebidas Alcoólicas/genética , Consumo de Bebidas Alcoólicas/metabolismo , Proteínas de Ligação ao GTP/genética , Fosfoglucomutase/genética , Proteínas Proto-Oncogênicas/genética , Acetaldeído/sangue , Adulto , Alelos , Concentração Alcoólica no Sangue , Feminino , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , República da Coreia/etnologiaRESUMO
The congenital disorders of glycosylation are a group of clinically and biochemically heterogeneous diseases characterized by multisystem involvement due to glycosylation defect of protein and lipid. Here we report a 49-year-old man with exercise-induced fatigue and pain of muscle, tachypnea, cleft palate and bifid uvula. Exercise induced elevation of serum creatine kinase (CK), ammonia and lactic acid was recorded. The abnormal levels of myoglobin, CK-MB and LDH as well as S-T elevation in electrocardiogram were observed in repeated hospitalization recordings. Electromyography showed myopathic damage. Repetitive nerve stimulation test of low rates showed decrement in the left deltoid muscle. He was identified with a novel homozygous frameshift variant in Phosphoglucomutase type 1 gene (c.405delT p.N135Kfs*9) by whole exome sequencing. Muscle biopsy exhibited minimal variation in fiber size without abnormal glycogen accumulation. Compared with controls', the patient's sample showed no signal at â¼61â¯kDa using N- or C-terminus antibody of Phosphoglucomutase type 1 in western blotting. A signal at â¼20â¯kDa was detected in patient using N-terminus antibody. Immunofluorescence revealed trace expression of C-terminus and a much lower expression of N-terminus on the sarcolemma than normal. Our findings indicate that c.405delT encodes a truncated protein with abnormal distribution and expression in skeletal muscle. In conclusion, genes associated with congenital disorders of glycosylation should be analyzed in patients with maxillofacial dysplasia, exertional weakness, cardiac involvement and exercise-induced-ammoniemia, without glycogen storage in skeletal muscle.
Assuntos
Defeitos Congênitos da Glicosilação , Fosfoglucomutase/genética , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Defeitos Congênitos da Glicosilação/fisiopatologia , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Sequenciamento do ExomaRESUMO
Helicobacter pylori growth requirements is a prerequisite to invade gastric epithelium and the process of injury to gastric cells will eventually lead to gastric cancer. The aim of this study is to investigate the effect of iron challenge on the expression of genes involved in iron homeostasis. The presence of Phosphoglucosamine mutase (glmM), cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) genes and mRNA expression of Iron Regulatory Protein 2 (IRP2), Transferrin Receptor (TFRC) and Ferritin Light Chain (FTL) genes in samples of 28 normal gastric mucosa, 33 chronic gastritis, 29 gastritis with intestinal metaplasia, 29 intestinal type adenocarcinoma patients were examined by real-time PCR. Immunohistochemistry was used to analyze cellular localization and protein levels. In the all H. pylori positive tissues, particularly in the basal regions of foveolar cells, TFRC was overexpressed (Pâ¯<â¯0.05), and regardless of the H. pylori infection, FTL was overexpressed in all patient, exclusively in metaplastic glandular cells (Pâ¯<â¯0.05). Furthermore, overexpression of IRP2 was associated with H. pylori positive chronic gastritis and intestinal metaplasia (Pâ¯<â¯0.05). Our findings confirm the role of transferrin receptor in H. pylori attachment into the gastric mucosa to capture iron. Overexpression of FTL gene in metaplastic cells could be considered as a research background to investigate the role of this gene in the differentiation of gastric cells into intestinal metaplasia. In addition, this gene could be suggested as a diagnostic marker to be included among the other markers routinely performed by clinical diagnostic laboratories.
Assuntos
Apoferritinas/metabolismo , Biomarcadores , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Helicobacter pylori/patogenicidade , Metaplasia/metabolismo , Receptores da Transferrina/metabolismo , Adenocarcinoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Apoferritinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Gastrite/patologia , Gastrite Atrófica/patologia , Expressão Gênica , Infecções por Helicobacter/patologia , Humanos , Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Masculino , Metaplasia/diagnóstico , Pessoa de Meia-Idade , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , RNA Mensageiro/biossíntese , Neoplasias Gástricas/patologia , Adulto JovemRESUMO
BACKGROUND: Leishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing. RESULTS: Partial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples. CONCLUSIONS: A HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated.
Assuntos
Leishmania infantum/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Polimorfismo Genético/genética , Genótipo , Glucose-6-Fosfato Isomerase/genética , Glucosefosfato Desidrogenase/genética , Proteínas de Helminto/genética , Humanos , Isocitrato Desidrogenase/genética , Isoenzimas/genética , Leishmania infantum/enzimologia , Leishmania infantum/isolamento & purificação , Manose-6-Fosfato Isomerase/genética , Fosfoglucomutase/genética , Fosfogluconato Desidrogenase/genética , Filogenia , Análise de Sequência de DNARESUMO
Glycogen metabolism commonly altered in cancer is just beginning to be understood. Phosphoglucomutase 1 (PGM1), the first enzyme in glycogenesis that catalyzes the reversible conversion between glucose 1-phosphate (G-1-P) and glucose 6-phosphate (G-6-P), participates in both the breakdown and synthesis of glycogen. Here, we show that PGM1 is down-regulated in hepatocellular carcinoma (HCC), which is associated with the malignancy and poor prognosis of HCC. Decreased PGM1 expression obstructed glycogenesis pathway, which leads to the increased flow of glucose into glycolysis, thereby promoting tumor cell proliferation and HCC development. The loss of forkhead box protein J2 (FOXJ2), at least partly due to low genomic copy number in HCC, releases cellular nucleic acid-binding protein (CNBP), a nucleic acid chaperon, to bind to and promote G-quadruplex formation in PGM1 promoter and therefore decreases PGM1 expression. In addition, integrated analyses of PGM1 and FOXJ2 expression provide a better prediction for the malignance and prognosis of HCC. This study establishes a tumor-suppressive role of PGM1 by regulating glucose trafficking and uncovers a novel regulatory mechanism of PGM1 expression.
Assuntos
Carcinoma Hepatocelular/metabolismo , Glucose/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfoglucomutase/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosfoglucomutase/deficiência , Fosfoglucomutase/genética , Prognóstico , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. CASE PRESENTATION: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. CONCLUSIONS: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.
Assuntos
Síndromes de Imunodeficiência/genética , Mutação , Fosfoglucomutase/genética , Sítios de Splice de RNA/genética , Pré-Escolar , Evolução Fatal , Feminino , Homozigoto , Humanos , Fosfoglucomutase/metabolismo , RNA Mensageiro/metabolismoRESUMO
The proinflammatory cytokines of TNF-α and IL-1ß have been reported to be increased in gastric mucosal surfaces in people with Helicobacter pylori infection. Accordingly, this study was conducted to investigate the relationship between the presence of H. pylori genes and the serum oscillations of these cytokines. In this study, DNA was first extracted from the stool samples of infected individuals and used as DNA template to investigate the presence of glmM and 16S rRNA genes in PCR. The ELISA assay was employed to examine serum levels of TNF-α and IL-1ß cytokines. According to statistical analysis, there was a significant correlation between the presence of glmM and 16S rRNA genes in the stool samples of infected persons and the serum oscillations of TNF-α and IL-1ß cytokines. At the end of study and analysis of the data in case group with HPSAg+, 47.6% of the glmM gene and 23.6% of the 16S rRNA gene were positive. In addition, a significant correlation was observed between the presence of glmM and 16S rRNA genes in the stool specimens of infected individuals and the serum levels of TNF-α and IL-1ß cytokines (p < 0.05). Considering the results, it can be concluded that fluctuations in the amount of HPSA, TNF-α, and IL-1ß in H. pylori infection depend on the presence of glmM and 16S rRNA genes. The presence of glmM and 16S rRNA in the stool sample increases by boosting the response level to stool antigen (HPSA), IL-1ß, and TNF-α, suggesting the prognosis of the disease with a bacterial virulence form using stool tests.