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1.
Trends Biochem Sci ; 45(3): 228-243, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31473074

RESUMO

Hundreds of metabolic enzymes work together smoothly in a cell. These enzymes are highly specific. Nevertheless, under physiological conditions, many perform side-reactions at low rates, producing potentially toxic side-products. An increasing number of metabolite repair enzymes are being discovered that serve to eliminate these noncanonical metabolites. Some of these enzymes are extraordinarily conserved, and their deficiency can lead to diseases in humans or embryonic lethality in mice, indicating their central role in cellular metabolism. We discuss how metabolite repair enzymes eliminate glycolytic side-products and prevent negative interference within and beyond this core metabolic pathway. Extrapolating from the number of metabolite repair enzymes involved in glycolysis, hundreds more likely remain to be discovered that protect a wide range of metabolic pathways.


Assuntos
Enzimas/metabolismo , Animais , Glicólise , Humanos , Camundongos
2.
Am J Hum Genet ; 108(6): 1151-1160, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33979636

RESUMO

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.


Assuntos
Glucose-6-Fosfato/análogos & derivados , Mutação , Transtornos do Neurodesenvolvimento/patologia , Fosfotransferases/genética , Alelos , Criança , Pré-Escolar , Feminino , Glucose-6-Fosfato/biossíntese , Glicosilação , Humanos , Masculino , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Linhagem
3.
Mol Genet Metab ; 141(3): 108144, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38277989

RESUMO

Glycogen storage disease type Ib (GSD Ib, biallelic variants in SLC37A4) is a rare disorder of glycogen metabolism complicated by neutropenia/neutrophil dysfunction. Since 2019, the SGLT2-inhibitor empagliflozin has provided a mechanism-based treatment option for the symptoms caused by neutropenia/neutrophil dysfunction (e.g. mucosal lesions, inflammatory bowel disease). Because of the rarity of GSD Ib, the published evidence on safety and efficacy of empagliflozin is still limited and does not allow to develop evidence-based guidelines. Here, an international group of experts provides 14 best practice consensus treatment recommendations based on expert practice and review of the published evidence. We recommend to start empagliflozin in all GSD Ib individuals with clinical or laboratory signs related to neutropenia/neutrophil dysfunction with a dose of 0.3-0.4 mg/kg/d given as a single dose in the morning. Treatment can be started in an outpatient setting. The dose should be adapted to the weight and in case of inadequate clinical treatment response or side effects. We strongly recommend to pause empagliflozin immediately in case of threatening dehydration and before planned longer surgeries. Discontinuation of G-CSF therapy should be attempted in all individuals. If available, 1,5-AG should be monitored. Individuals who have previously not tolerated starches should be encouraged to make a new attempt to introduce starch in their diet after initiation of empagliflozin treatment. We advise to monitor certain safety and efficacy parameters and recommend continuous, alternatively frequent glucose measurements during the introduction of empagliflozin. We provide specific recommendations for special circumstances like pregnancy and liver transplantation.


Assuntos
Compostos Benzidrílicos , Glucosídeos , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Humanos , Neutrófilos/metabolismo , Consenso , Doença de Depósito de Glicogênio Tipo I/complicações , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/genética , Neutropenia/tratamento farmacológico , Neutropenia/etiologia , Proteínas de Transporte de Monossacarídeos , Antiporters/metabolismo
4.
Mol Genet Metab ; 142(2): 108486, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38733639

RESUMO

Empagliflozin has been successfully repurposed for treating neutropenia and neutrophil dysfunction in patients with glycogen storage disease type 1b (GSD 1b), however, data in infants are missing. We report on efficacy and safety of empagliflozin in infants with GSD 1b. This is an international retrospective case series on 21 GSD 1b infants treated with empagliflozin (total treatment time 20.6 years). Before starting empagliflozin (at a median age of 8.1 months with a median dose of 0.3 mg/kg/day) 12 patients had clinical signs and symptoms of neutrophil dysfunction. Six of these previously symptomatic patients had no further neutropenia/neutrophil dysfunction-associated findings on empagliflozin. Eight patients had no signs and symptoms of neutropenia/neutrophil dysfunction before start and during empagliflozin treatment. One previously asymptomatic individual with a horseshoe kidney developed a central line infection with pyelonephritis and urosepsis during empagliflozin treatment. Of the 10 patients who were treated with G-CSF before starting empagliflozin, this was stopped in four and decreased in another four. Eleven individuals were never treated with G-CSF. While in 17 patients glucose homeostasis remained stable on empagliflozin, four showed glucose homeostasis instability in the introductory phase. In 17 patients, no other side effects were reported, while genital (n = 2) or oral (n = 1) candidiasis and skin infection (n = 1) were reported in the remaining four. Empagliflozin had a good effect on neutropenia/neutrophil dysfunction-related signs and symptoms and a favourable safety profile in infants with GSD 1b and therefore qualifies for further exploration as first line treatment.


Assuntos
Compostos Benzidrílicos , Glucosídeos , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Neutrófilos , Humanos , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/complicações , Neutropenia/tratamento farmacológico , Masculino , Feminino , Lactente , Compostos Benzidrílicos/uso terapêutico , Compostos Benzidrílicos/administração & dosagem , Estudos Retrospectivos , Neutrófilos/efeitos dos fármacos , Glucosídeos/uso terapêutico , Glucosídeos/farmacologia , Glucosídeos/administração & dosagem , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Resultado do Tratamento , Fator Estimulador de Colônias de Granulócitos/uso terapêutico
5.
Cell Mol Life Sci ; 80(9): 259, 2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37594549

RESUMO

Neutropenia and neutrophil dysfunction in glycogen storage disease type 1b (GSD1b) and severe congenital neutropenia type 4 (SCN4), associated with deficiencies of the glucose-6-phosphate transporter (G6PT/SLC37A4) and the phosphatase G6PC3, respectively, are the result of the accumulation of 1,5-anhydroglucitol-6-phosphate in neutrophils. This is an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol in blood. 1,5-AG is presumed to be reabsorbed in the kidney by a sodium-dependent-transporter of uncertain identity, possibly SGLT4/SLC5A9 or SGLT5/SLC5A10. Lowering blood 1,5-AG with an SGLT2-inhibitor greatly improved neutrophil counts and function in G6PC3-deficient and GSD1b patients. Yet, this effect is most likely mediated indirectly, through the inhibition of the renal 1,5-AG transporter by glucose, when its concentration rises in the renal tubule following inhibition of SGLT2. To identify the 1,5-AG transporter, both human and mouse SGLT4 and SGLT5 were expressed in HEK293T cells and transport measurements were performed with radiolabelled compounds. We found that SGLT5 is a better carrier for 1,5-AG than for mannose, while the opposite is true for human SGLT4. Heterozygous variants in SGLT5, associated with a low level of blood 1,5-AG in humans cause a 50-100% reduction in 1,5-AG transport activity tested in model cell lines, indicating that SGLT5 is the predominant kidney 1,5-AG transporter. These and other findings led to the conclusion that (1) SGLT5 is the main renal transporter of 1,5-AG; (2) frequent heterozygous mutations (allelic frequency > 1%) in SGLT5 lower blood 1,5-AG, favourably influencing neutropenia in G6PC3 or G6PT deficiency; (3) the effect of SGLT2-inhibitors on blood 1,5-AG level is largely indirect; (4) specific SGLT5-inhibitors would be more efficient to treat these neutropenias than SGLT2-inhibitors.


Assuntos
Neutropenia , Animais , Humanos , Camundongos , Antiporters , Células HEK293 , Rim , Proteínas de Membrana Transportadoras , Proteínas de Transporte de Monossacarídeos/genética , Neutropenia/genética , Transportador 2 de Glucose-Sódio/genética
6.
Mol Genet Metab ; 140(3): 107712, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38353183

RESUMO

Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3, respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra-individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients' clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients' follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3-deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors.


Assuntos
Desoxiglucose , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Inibidores do Transportador 2 de Sódio-Glicose , Criança , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/complicações , Neutropenia/genética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Monoéster Fosfórico Hidrolases , Proteínas de Transporte de Monossacarídeos , Antiporters
7.
Cell Mol Life Sci ; 79(8): 421, 2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35834009

RESUMO

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.


Assuntos
Aminoácidos , Transaminases , Citosol/metabolismo , Humanos , Cinética , Especificidade por Substrato , Açúcares , Transaminases/metabolismo
8.
J Biol Chem ; 296: 100699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895133

RESUMO

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico , Estereoisomerismo
9.
Blood ; 136(9): 1033-1043, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32294159

RESUMO

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)-deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Glucosídeos/uso terapêutico , Doença de Depósito de Glicogênio Tipo I/complicações , Hexosefosfatos/sangue , Neutropenia/tratamento farmacológico , Neutrófilos/patologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Compostos Benzidrílicos/efeitos adversos , Glicemia/análise , Quimiotaxia de Leucócito/efeitos dos fármacos , Pré-Escolar , Reposicionamento de Medicamentos , Resistência a Medicamentos , Feminino , Glucosídeos/efeitos adversos , Doença de Depósito de Glicogênio Tipo I/sangue , Doença de Depósito de Glicogênio Tipo I/imunologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Granulócitos/química , Humanos , Recém-Nascido , Proteína 2 de Membrana Associada ao Lisossomo/sangue , Masculino , Neutropenia/sangue , Uso Off-Label , Explosão Respiratória/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Adulto Jovem
10.
J Inherit Metab Dis ; 45(4): 759-768, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35506446

RESUMO

Neutropenia and neutrophil dysfunction found in deficiencies in G6PC3 and in the glucose-6-phosphate transporter (G6PT/SLC37A4) are due to accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), an inhibitor of hexokinase made from 1,5-anhydroglucitol (1,5-AG), an abundant polyol present in blood. Lowering blood 1,5-AG with an SGLT2 inhibitor greatly improved neutrophil counts and function in G6PC3-deficient mice and in patients with G6PT-deficiency. We evaluate this treatment in two G6PC3-deficient children. While neutropenia was severe in one child (PT1), which was dependent on granulocyte cololony-stimulating factor (GCSF), it was significantly milder in the other one (PT2), which had low blood 1,5-AG levels and only required GCSF during severe infections. Treatment with the SGLT2-inhibitor empagliflozin decreased 1,5-AG in blood and 1,5-AG6P in neutrophils and improved (PT1) or normalized (PT2) neutrophil counts, allowing to stop GCSF. On empagliflozin, both children remained infection-free (>1 year - PT2; >2 years - PT1) and no side effects were reported. Remarkably, sequencing of SGLT5, the gene encoding the putative renal transporter for 1,5-AG, disclosed a rare heterozygous missense mutation in PT2, replacing the extremely conserved Arg401 by a histidine. The higher urinary clearance of 1,5-AG explains the more benign neutropenia and the outstanding response to empagliflozin treatment found in this child. Our data shows that SGLT2 inhibitors are an excellent alternative to treat the neutropenia present in G6PC3-deficiency.


Assuntos
Doença de Depósito de Glicogênio Tipo I , Neutropenia , Proteínas de Transporte de Sódio-Glucose/metabolismo , Animais , Antiporters/genética , Compostos Benzidrílicos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Glucosídeos/uso terapêutico , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/genética , Humanos , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Neutropenia/tratamento farmacológico , Neutropenia/genética , Monoéster Fosfórico Hidrolases/genética
11.
Proc Natl Acad Sci U S A ; 116(4): 1241-1250, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30626647

RESUMO

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-KM hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-KM hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.


Assuntos
Antiporters/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Neutropenia/metabolismo , Fosforilação/fisiologia , Animais , Morte Celular/fisiologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Ratos Wistar
12.
J Clin Immunol ; 41(5): 958-966, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33534079

RESUMO

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 , Masculino
13.
J Inherit Metab Dis ; 44(5): 1215-1225, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33973257

RESUMO

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short-chain acyl-CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl-CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss-of-function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven.


Assuntos
Acil-CoA Desidrogenase/deficiência , Variação Genética , Erros Inatos do Metabolismo Lipídico/genética , Malonatos/metabolismo , Enzima Bifuncional do Peroxissomo/genética , Acil-CoA Desidrogenase/genética , Alelos , Frequência do Gene , Genótipo , Células HEK293 , Humanos , Deficiência Múltipla de Acil Coenzima A Desidrogenase
14.
Anal Biochem ; 593: 113595, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31987861

RESUMO

Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.


Assuntos
Ensaios Enzimáticos/métodos , Enzimas/química , Artefatos , Cinética , Ornitina-Oxo-Ácido Transaminase/química , Especificidade por Substrato
15.
J Inherit Metab Dis ; 43(1): 14-24, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31691304

RESUMO

It is traditionally assumed that enzymes of intermediary metabolism are extremely specific and that this is sufficient to prevent the production of useless and/or toxic side-products. Recent work indicates that this statement is not entirely correct. In reality, enzymes are not strictly specific, they often display weak side activities on intracellular metabolites (substrate promiscuity) that resemble their physiological substrate or slowly catalyse abnormal reactions on their physiological substrate (catalytic promiscuity). They thereby produce non-classical metabolites that are not efficiently metabolised by conventional enzymes. In an increasing number of cases, metabolite repair enzymes are being discovered that serve to eliminate these non-classical metabolites and prevent their accumulation. Metabolite repair enzymes also eliminate non-classical metabolites that are formed through spontaneous (ie, not enzyme-catalysed) reactions. Importantly, genetic deficiencies in several metabolite repair enzymes lead to 'inborn errors of metabolite repair', such as L-2-hydroxyglutaric aciduria, D-2-hydroxyglutaric aciduria, 'ubiquitous glucose-6-phosphatase' (G6PC3) deficiency, the neutropenia present in Glycogen Storage Disease type Ib or defects in the enzymes that repair the hydrated forms of NADH or NADPH. Metabolite repair defects may be difficult to identify as such, because the mutated enzymes are non-classical enzymes that act on non-classical metabolites, which in some cases accumulate only inside the cells, and at rather low, yet toxic, concentrations. It is therefore likely that many additional metabolite repair enzymes remain to be discovered and that many diseases of metabolite repair still await elucidation.


Assuntos
Enzimas/metabolismo , Enzimas/fisiologia , Redes e Vias Metabólicas/fisiologia , Erros Inatos do Metabolismo/prevenção & controle , Metabolismo/fisiologia , Encefalopatias Metabólicas Congênitas/metabolismo , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Neutropenia/metabolismo
16.
Biochem J ; 476(16): 2427-2447, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31416829

RESUMO

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function.


Assuntos
Acil Coenzima A/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/biossíntese , Células 3T3-L1 , Acil Coenzima A/genética , Animais , Descarboxilação , Ácido Graxo Sintase Tipo I/genética , Ácidos Graxos/genética , Camundongos
17.
Proc Natl Acad Sci U S A ; 114(16): E3233-E3242, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28373563

RESUMO

The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.


Assuntos
Aminoidrolases/metabolismo , Glutationa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Aminoidrolases/fisiologia , Animais , Desaminação , Humanos , Hidrólise , Camundongos , Camundongos Knockout , Especificidade por Substrato
18.
Nat Chem Biol ; 12(8): 601-7, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27294321

RESUMO

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.


Assuntos
Glicolatos/metabolismo , Glicólise , Lactatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Açúcares Ácidos/metabolismo , Glicolatos/química , Glicolatos/toxicidade , Glicólise/efeitos dos fármacos , Células HCT116 , Humanos , Lactatos/química , Lactatos/toxicidade , Via de Pentose Fosfato/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/deficiência , Fosforilação , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Açúcares Ácidos/química , Açúcares Ácidos/toxicidade
19.
J Physiol ; 594(17): 4849-63, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27062388

RESUMO

KEY POINTS: Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that ß-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of ß-alanine, which is in turn controlled by degradation of ß-alanine in liver and kidney. Chronic oral ß-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the ß-alanine supplementation protocol is so inefficient, by demonstrating that exogenous ß-alanine can be effectively routed toward oxidation. ABSTRACT: The metabolic fate of orally ingested ß-alanine is largely unknown. Chronic ß-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (ß-alanyl-l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested ß-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two ß-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate ß-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on ß-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating ß-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating ß-alanine levels, which are suppressed by hepatic and renal ß-alanine transamination upon oral ß-alanine intake.


Assuntos
Anserina/metabolismo , Carnosina/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Transaminases/metabolismo , beta-Alanina/metabolismo , Ácido Amino-Oxiacético/farmacologia , Animais , Encéfalo/metabolismo , Inibidores Enzimáticos/farmacologia , GABAérgicos/farmacologia , Células HEK293 , Homeostase , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transaminases/antagonistas & inibidores , Transaminases/genética , Vigabatrina/farmacologia , beta-Alanina/sangue , beta-Alanina/urina
20.
Cell Mol Life Sci ; 72(23): 4633-51, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26077601

RESUMO

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.


Assuntos
Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Linhagem Celular , Membrana Eritrocítica/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Microdomínios da Membrana/química , Camundongos , Mioblastos/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo , Temperatura
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