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1.
Int J Mol Sci ; 23(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36142650

RESUMO

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.


Assuntos
Yarrowia , Carbono/metabolismo , Diacilglicerol Colinofosfotransferase/metabolismo , Diacilglicerol Quinase/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Glicerol Quinase/metabolismo , Lipossomos/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatidil-N-Metiletanolamina N-Metiltransferase/metabolismo , Fosfatidilcolinas/metabolismo , Triglicerídeos/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
2.
Lipids ; 57(6): 313-325, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36098349

RESUMO

Although it is well established that glucocorticoids inactivate thermogenesis and promote lipid accumulation in interscapular brown adipose tissue (IBAT), the underlying mechanisms remain unknown. We found that dexamethasone treatment (1 mg/kg) for 7 days in rats decreased the IBAT thermogenic activity, evidenced by its lower responsiveness to noradrenaline injection associated with reduced content of mitochondrial proteins, respiratory chain protein complexes, noradrenaline, and the ß3 -adrenergic receptor. In parallel, to understand better how dexamethasone increases IBAT lipid content, we also investigated the activity of the ATP citrate lyase (ACL), a key enzyme of de novo fatty acid synthesis, glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, and the three glycerol-3-P generating pathways: (1) glycolysis, estimated by 2-deoxyglucose uptake, (2) glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase activity and pyruvate incorporation into triacylglycerol-glycerol, and (3) direct phosphorylation of glycerol, investigated by the content and activity of glycerokinase. Dexamethasone increased the mass and the lipid content of IBAT as well as plasma levels of glucose, insulin, non-esterified fatty acid, and glycerol. Furthermore, dexamethasone increased ACL and G6PD activities (79% and 48%, respectively). Despite promoting a decrease in the incorporation of U-[14 C]-glycerol into triacylglycerol (~54%), dexamethasone increased the content (~55%) and activity (~41%) of glycerokinase without affecting glucose uptake or glyceroneogenesis. Our data suggest that glucocorticoid administration reduces IBAT thermogenesis through sympathetic inactivation and stimulates glycerokinase activity and content, contributing to increased generation of glycerol-3-P, which is mostly used to esterify fatty acid and increase triacylglycerol content promoting IBAT whitening.


Assuntos
Tecido Adiposo Marrom , Glicerol Quinase , Animais , Ratos , Tecido Adiposo Marrom/metabolismo , Glicerol Quinase/metabolismo , Glucocorticoides , Glicerol , Ratos Wistar , Termogênese , Triglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Dexametasona/metabolismo , Norepinefrina , Tecido Adiposo/metabolismo
3.
BMC Pediatr ; 22(1): 517, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050749

RESUMO

BACKGROUND: Xp21 contiguous gene deletion syndrome is a rare genetic metabolic disorder with poor prognosis in infants, involving deletions of one or more genes in Xp21. When deletions of adrenal hypoplasia (AHC), Duchenne muscular dystrophy (DMD), and chronic granulomatosis (CGD) loci are included, complex glycerol kinase deficiency (CGKD) can be diagnosed. We present a case of CGKD that was initially misdiagnosed and died during treatment in our hospital in terms of improving our understanding of the clinical features and diagnosis of this disease, as well as highlighting the need for more precise dosing of corticosteroid replacement therapy. CASE PRESENTATION: A 48-day-old full-term male infant was transferred to our medical center with global growth delay and persistent vomiting. Routine laboratory tests revealed hyperkalemia, hyponatremia, and a high level of creatine kinase. The initial diagnosis was adrenal cortical hyperplasia (ACH), then revised to adrenocortical insufficiency with a normal level of ACTH detected. After supplementing the routine lipid test and urinary glycerol test, CGKD was diagnosed clinically due to positive triglyceridemia and urinary glycerol, and the follow-up gene screening further confirmed the diagnosis. The boy kept thriving after corticosteroid replacement and salt supplementation. While levels of serum ACTH and cortisol decreased and remained low after corticosteroid replacement was administered. The patient died of acute type 2 respiratory failure and hypoglycemia after an acute upper respiratory tract infection, which may be the result of adrenal crisis after infection. Infants with CGKD have a poor prognosis, so physicians should administer regular follow-ups, and parents counseling during treatment to improve the survival of patients. CONCLUSIONS: Overall, CGKD, although rare, cannot be easily excluded in children with persistent vomiting. Extensive blood tests can help to detect abnormal indicators. Adrenal crisis needs to be avoided as much as possible during corticosteroid replacement therapy.


Assuntos
Insuficiência Adrenal , Glicerol Quinase , Insuficiência Adrenal/diagnóstico , Insuficiência Adrenal/genética , Hormônio Adrenocorticotrópico , Criança , China , Diagnóstico Tardio , Glicerol , Glicerol Quinase/genética , Humanos , Hipoadrenocorticismo Familiar , Lactente , Masculino , Vômito
4.
Biotechnol Lett ; 44(9): 1051-1061, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35922648

RESUMO

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.


Assuntos
Bacillus subtilis , Glicerol Quinase , Cromossomos/metabolismo , Escherichia coli/metabolismo , Glicerol/metabolismo , Glicerol Quinase/química , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Mutagênese Sítio-Dirigida
5.
Appl Microbiol Biotechnol ; 106(7): 2541-2555, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35325274

RESUMO

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.


Assuntos
Aquaporinas , Cupriavidus necator , Proteínas de Escherichia coli , Aquaporinas/metabolismo , Dióxido de Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicerol/metabolismo , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
6.
BMJ Case Rep ; 15(3)2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35292548

RESUMO

High triglyceride in a serum sample with no apparent visible lipaemia is a confusing laboratory condition. This condition of apparent hypertriglyceridaemia might be an asymptomatic condition of pseudo-hypertriglyceridaemia which is seen in individuals with glycerol kinase deficiency. Glycerol is increased in glycerol kinase deficiency, therefore biochemical analysers that measure glycerol to estimate triglyceride report high triglyceride values. Clinicians are often unaware of this laboratory condition; as a result, patients are subjected to aggressive hypolipidaemic drugs and lifestyle changes, and these measures turn ineffective to lower triglyceride levels. In this report, a case of a 50-year-old Nepalese male is presented with an apparent hypertriglyceridaemia refractory to various hypolipidaemic drugs for the last 13 years until a correct diagnosis of his condition was made.


Assuntos
Hiperlipidemias , Hipertrigliceridemia , Erros de Diagnóstico , Glicerol Quinase , Humanos , Hipertrigliceridemia/diagnóstico , Hipertrigliceridemia/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Triglicerídeos
7.
Sci Rep ; 12(1): 4129, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260770

RESUMO

The fall armyworm (FAW), Spodoptera frugiperda, is native to the tropical and subtropical areas of the American continent and is one of the world's most destructive insect pests and invaded Africa and spread to most of Asia in two years. Glycerol is generally used as a cryoprotectant for overwintering insects in cold areas. In many studies, the increase in glycerol as a main rapid cold hardening (RCH) factor and enhancing the supercooling point was revealed at low temperatures. There are two genes, including glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), that were identified as being associated with the glycerol synthesis pathway. In this study, one GPDH and two GK sequences (GK1 and GK2) were extracted from FAW transcriptome analysis. RNA interference (RNAi) specific to GPDH or GK1 and GK2 exhibited a significant down-regulation at the mRNA level as well as a reduction in survival rate when the RNAi-treated of FAW larvae post a RCH treatment. Following a cold period, an increase in glycerol accumulation was detected utilizing high-pressure liquid chromatography and colorimetric analysis of glycerol quantity in RCH treated hemolymph of FAW larvae. This research suggests that GPDH and GK isozymes are linked to the production of a high quantity of glycerol as an RCH factor, and glycerol as main cryoprotectant plays an important role in survival throughout the cold period in this quarantine pest studied.


Assuntos
Glicerol , Mariposas , Animais , Crioprotetores , Glicerol/metabolismo , Glicerol Quinase/genética , Glicerolfosfato Desidrogenase/metabolismo , Larva/fisiologia , Mariposas/metabolismo , Spodoptera/genética , Spodoptera/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-35105298

RESUMO

BACKGROUND: Chromosome Xp21 deletion syndrome is a rare X-linked recessive defect that occurs as a result of multiple gene deletions, including Glycerol kinase (GK) and its neighboring genes, dystrophin, which causes Duchenne muscular dystrophy (DMD), and NR0B1, which causes congenital adrenal hypoplasia (CAHhttps://www.omim.org/entry/300200). Patients usually present with glycerol kinase deficiency, congenital adrenal hypoplasia, Duchenne muscular dystrophy, hyperglycerolemia, and glyceroluria, associated with DMD and/or CAH, growth failure, myopathy, osteoporosis, mental retardation, and psychomotor retardation. CASE PRESENTATION: Herein, we report a 3-year- old boy from Iraq who had bloody diarrhea, food intolerance and abdominal cramp, adrenal insufficiency, recurrent fevers, tuberculosis (TB) infection, cervical abscess, oral thrush, cervical and mediastinal lymphadenopathies, developmental delay, and undescended testis. His parents are non-consanguine and had no family history of diseases. Next generation sequencing demonstrated a hemizygote deletion in chromosome X. CONCLUSION: Loss of a large part of the X-chromosome most likely can explain the clinical findings of this patient. Contiguous gene deletion syndrome in Xp21 should be considered after diagnosing adrenal insufficiency to treat metabolic complications efficiently.


Assuntos
Insuficiência Adrenal , Distrofia Muscular de Duchenne , Pré-Escolar , Glicerol Quinase , Humanos , Hipoadrenocorticismo Familiar , Masculino , Síndrome , Cromossomo X
9.
PLoS Negl Trop Dis ; 15(12): e0009985, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34919562

RESUMO

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Glicerol Quinase/sangue , Lisofosfolipase/sangue , Proteínas de Protozoários/sangue , Testes Sorológicos/métodos , Trypanosoma/imunologia , Tripanossomíase Bovina/diagnóstico , Animais , Bovinos , Glicerol Quinase/genética , Glicerol Quinase/imunologia , Lisofosfolipase/genética , Lisofosfolipase/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma/classificação , Trypanosoma/enzimologia , Trypanosoma/genética , Tripanossomíase Bovina/sangue , Tripanossomíase Bovina/parasitologia
10.
Pediatr Endocrinol Diabetes Metab ; 27(3): 227-231, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34743506

RESUMO

Complex glycerol kinase deficiency (CGKD) is a rare genetic syndrome which belongs to the group of contiguous gene syndromes and is caused by microdeletion of genes located in Xp21. Patients with CGKD present with features characteristic for adrenal hypoplasia, glycerol kinase deficiency, Duchenne muscular dystrophy and sometimes intellectual disability. We present a long-term follow-up of two unrelated boys with molecular diagnosis of complex glycerol kinase deficiency. Genetic examinations in both patients revealed a deletion on Xp21 chromosome including complete deletion of NR0B1 and GK genes. Additionally in patient 2 IL1RAPL1 genes were deleted. In separate MLPA test DMD gene deletion was diagnosed in both patients as follow: in patient 1 whole gene while in patient 2 the C-terminal region of DMD was deleted. Although the first symptom in both was salt loss syndrome, the course of the disease was different for them. We share our experience resulting from the opportunity of caring for patients with this rare disease from the beginning of their life to the end of pediatric care.


Assuntos
Glicerol Quinase , Criança , Seguimentos , Glicerol Quinase/genética , Humanos , Hipoadrenocorticismo Familiar , Masculino , Síndrome
11.
Ann Biol Clin (Paris) ; 79(5): 453-455, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726160

RESUMO

Currently, the determination of triglycerides (TG) uses an indirect method: the concentration of TG is assayed from the amount of free glycerol released by the hydrolysis of TG into fatty acids (FA) and glycerol. In general, the physiological glycerol before hydrolysis is very low. However, in some situations, glycerolaemia may be significantly elevated, due to the presence of exogenous or endogenous glycerol giving rise to (pseudo) hypertriglyceridemia. Here, we report a 48-year-old male patient with type 2 diabetes presenting with a pseudo-hypertriglyceridemia based on a pronounced hyperglycerolaemia. The hyperglycerolaemia was due to a deficiency of glycerol kinase (GKD). In conclusion, unexplained hypertriglyceridemia should raise the suspicion of hyperglycerolaemia, especially when it resists well-conducted fibrate treatment, with a non-lipemic serum and which is not associated with an increase in pre-beta lipoproteins or abnormal presence of chylomicrons.


Assuntos
Diabetes Mellitus Tipo 2 , Hiperlipidemias , Hipertrigliceridemia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Glicerol Quinase , Humanos , Hipertrigliceridemia/diagnóstico , Masculino , Pessoa de Meia-Idade , Triglicerídeos
12.
BMC Endocr Disord ; 21(1): 214, 2021 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-34689766

RESUMO

BACKGROUND: Contiguous gene deletion syndromes are rare genomic disorders caused by deletion of large segments of DNA resulting in co-occurrence of apparently unrelated multiple clinical phenotypes. We report a boy with contiguous gene deletion involving Xp21 genomic location. CASE PRESENTATION: A Sri Lankan boy with developmental delay and failure to thrive first presented at three years of age with hypovolaemia, hyperpigmentation and drowsiness. Investigations done at that time revealed hypoglycaemia, hyponatraemia, hyperkalaemia, low cortisol, low aldosterone, high ACTH and low 17-hydroxyprogesterone. He was diagnosed to have primary adrenal insufficiency. During follow-up at five years, he was noted to have progressive difficulty in walking, waddling gait, hypotonia, calf hypertrophy and positive Gower's sign. His creatine kinase was very high, and the electromyogram showed myopathy. Genetic analysis revealed hemizygous deletion involving the final 35 exons of the dystrophin gene confirming the diagnosis of Duchenne muscular dystrophy. Further investigations revealed pseudohypertriglyceridemia, large glycerol peak on urine organic acid analysis and hemizygous deletion of the glycerol kinase gene confirming glycerol kinase deficiency. Based on the presence of Duchenne muscular dystrophy, glycerol kinase deficiency and probable congenital adrenal hypoplasia along with genetic confirmation of deletions involving dystrophin and glycerol kinase genes, the diagnosis of Xp21 contiguous gene deletion syndrome was made. CONCLUSIONS: We report a child with contiguous gene deletion syndrome who was initially diagnosed as having isolated primary adrenal insufficiency probably due to congenital adrenal hypoplasia. Later he was confirmed to have Duchenne muscular dystrophy and glycerol kinase deficiency, as well. This case report highlights the importance of pre-emptive evaluation and identification of genetic defects when patients present with seemingly unrelated diseases that could aid in accurate diagnoses of contiguous gene deletion syndromes.


Assuntos
Glicerol Quinase/deficiência , Hipoadrenocorticismo Familiar/complicações , Distrofia Muscular de Duchenne/complicações , Pré-Escolar , Humanos , Hipoadrenocorticismo Familiar/metabolismo , Masculino
13.
Protein Sci ; 30(12): 2457-2473, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34655136

RESUMO

Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.


Assuntos
Adaptação Fisiológica/genética , Deutério/metabolismo , Escherichia coli K12/metabolismo , Marcação por Isótopo/métodos , Nêutrons , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Glicerol/metabolismo , Glicerol/farmacologia , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Mutação , Difração de Nêutrons , Canais de Potássio/genética , Canais de Potássio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Seleção Genética , Fator sigma/genética , Fator sigma/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequenciamento Completo do Genoma
14.
PLoS Biol ; 19(8): e3001359, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34388147

RESUMO

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.


Assuntos
Glicerol Quinase/metabolismo , Glicerol/metabolismo , Hexoquinase/metabolismo , Microcorpos/enzimologia , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular
15.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299193

RESUMO

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.


Assuntos
Citrato (si)-Sintase/metabolismo , Suplementos Nutricionais , Glicerol Quinase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Engenharia Metabólica/métodos , Yarrowia/fisiologia , Biomassa , Meios de Cultura , Ácidos Cetoglutáricos/isolamento & purificação
16.
Fungal Genet Biol ; 149: 103531, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33581364

RESUMO

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.


Assuntos
Aspergillus fumigatus/metabolismo , Glicerol Quinase/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Estresse Oxidativo/fisiologia , Aspergillus fumigatus/genética , Glucose/metabolismo , Glicerol/metabolismo , Glicerol Quinase/fisiologia , Glicerolfosfato Desidrogenase/biossíntese , Glicerofosfatos , Peróxido de Hidrogênio/metabolismo , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , Oxirredução , Fenótipo , Fosfatos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Clin Exp Pharmacol Physiol ; 48(4): 498-507, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33462866

RESUMO

Particulate matter (PM2.5) and cigarette smoke exposure are leading factors contributing to various diseases, especially respiratory diseases. The purpose of this research was to study the effects of PM2.5 and cigarette smoke on glycerol kinase 5 (GK5) expression and the possible mechanisms by which GK5 participates in lipid droplet (LD) synthesis in alveolar epithelial A549 cells. Real-time polymerase chain reaction (RT-PCR) and western blotting have been used for the detection of messenger RNA (mRNA) and protein expression respectively. GK5 overexpressing cells were established by lentivirus transfection, whereby lentiviral vectors deliver the gene into chromosomes, allowing stable expression. Affymetrix microarray analysis, a widely used tool for measuring genome-wide gene expression, has been used to explore differential gene expression profiles. A549 cells stimulated with PM2.5 and cigarette smoke extract (CSE) showed elevated GK5 expression in a dose-dependent manner. Transmission electron microscopy and oil red O staining were used to observe LDs in cells. Further, GK5 overexpressing cells showed increased LDs and upregulation of genes and proteins related to lipogenesis and lipid transportation. Affymetrix microarray analysis revealed that GK5 overexpression resulted in the differential expression of more than 109 genes, which were mainly involved in the regulation of cell death, cell survival, cellular movement and migration, and those involved in cellular growth and proliferation pathways. Overall, this study demonstrates that GK5 is upregulated during PM2.5 and cigarette smoke exposure and induces LD synthesis.


Assuntos
Glicerol Quinase , Material Particulado , Células A549 , Apoptose , Humanos , Gotículas Lipídicas , Doença Pulmonar Obstrutiva Crônica , Fumaça
18.
FASEB J ; 35(10): e21911, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34551152

RESUMO

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.


Assuntos
Proteínas de Transporte/genética , Citocinas/deficiência , Citocinas/genética , Deleção de Genes , Intolerância à Glucose/genética , Glicerol Quinase/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Animais , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Feminino , Glucose/biossíntese , Glucose/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Gravidez , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismo , Aumento de Peso/genética
19.
Microb Cell Fact ; 19(1): 227, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308214

RESUMO

BACKGROUND: A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. RESULTS: We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. CONCLUSION: The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Proteínas Recombinantes de Fusão/biossíntese , Asparaginase/genética , Asparaginase/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Transdução de Sinais , Estresse Fisiológico , Regulação para Cima , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética
20.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348713

RESUMO

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h-1. A GL3 derivative with a chromosomally integrated copy of fsaAA129S (GL4) grew with 0.05 h-1 on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created.


Assuntos
Aldeído Liases/genética , Vias Biossintéticas/genética , Di-Hidroxiacetona/biossíntese , Escherichia coli K12/enzimologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Glicerol/metabolismo , Alelos , Frutosefosfatos/metabolismo , Deleção de Genes , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glicerol Quinase/genética , Isoenzimas/genética , Via de Pentose Fosfato/genética , Fosfofrutoquinases/química , Fosfofrutoquinases/genética , Desidrogenase do Álcool de Açúcar/genética
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