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2.
Neurochem Int ; 139: 104816, 2020 10.
Article in English | MEDLINE | ID: mdl-32758590

ABSTRACT

Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. We herein established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons.


Subject(s)
Dopaminergic Neurons/enzymology , Parkinsonian Disorders/enzymology , Parkinsonian Disorders/genetics , Phosphoglycerate Kinase/antagonists & inhibitors , Phosphoglycerate Kinase/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Dopaminergic Neurons/pathology , Drosophila , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/deficiency , Drosophila Proteins/genetics , Humans , Parkinsonian Disorders/pathology , Phosphoglycerate Kinase/deficiency
3.
J Pediatr Hematol Oncol ; 42(4): e228-e230, 2020 05.
Article in English | MEDLINE | ID: mdl-30951021

ABSTRACT

Phosphoglycerate kinase (PGK) is glycolytic enzyme critical in the creation of adenosine triphosphate. Mutations in the gene for this enzyme, PGK1, are associated with PGK deficiency, which is characterized by neurologic symptoms, nonhereditary spherocytic hemolytic anemia, and myopathy. We present a 20-year-old male with a novel c.461T>C (p.L154P) PGK1 mutation and clinical disease complicated by anemia and neurological symptoms. There is no recommended treatment for PGK deficiency. Because of our patient's advanced disease progression, we initiated serial blood transfusions and report significant subjective improvement in the patient's physical condition before his passing from PGK deficiency-related complications.


Subject(s)
Blood Transfusion , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/therapy , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/therapy , Phosphoglycerate Kinase/deficiency , Point Mutation , Adult , Humans , Male , Phosphoglycerate Kinase/genetics
4.
Genes (Basel) ; 10(10)2019 10 10.
Article in English | MEDLINE | ID: mdl-31658606

ABSTRACT

Phosphoglycerate kinase (PGK)1 deficiency is an X-linked inherited disease associated with different clinical presentations, sometimes as myopathic affectation without hemolytic anemia. We present a 40-year-old male with a mild psychomotor delay and mild mental retardation, who developed progressive exercise intolerance, cramps and sporadic episodes of rhabdomyolysis but no hematological features. A genetic study was carried out by a next-generation sequencing (NGS) panel of 32 genes associated with inherited metabolic myopathies. We identified a missense variant in the PGK1 gene c.1114G > A (p.Gly372Ser) located in the last nucleotide of exon 9. cDNA studies demonstrated abnormalities in mRNA splicing because this change abolishes the exon 9 donor site. This novel variant is the first variant associated with a myopathic form of PGK1 deficiency in the Spanish population.


Subject(s)
Genetic Diseases, X-Linked/genetics , Metabolism, Inborn Errors/genetics , Mutation, Missense , Phosphoglycerate Kinase/genetics , Adult , Cells, Cultured , Genetic Diseases, X-Linked/pathology , Humans , Male , Metabolism, Inborn Errors/pathology , Phosphoglycerate Kinase/deficiency , Phosphoglycerate Kinase/metabolism , RNA Splicing , Spain
5.
Pediatr Hematol Oncol ; 36(5): 302-308, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31424298

ABSTRACT

The human phosphoglycerate kinase-1 enzyme is the first of two energy generating steps in the glycolysis. Since its discovery in 1968, many pathologically mutated forms of PGK1 have been described. PGK1 is expressed in all tissues. The clinical manifestations of PGK1 deficiency are some combination of anemia, central nervous system and/or musculoskeletal manifestations. We describe a case of PGK1 in an African-American child, which to our knowledge, has never been described to date. The manifestations of PGK1-Detroit (c.1105A > C (p.Thr369Pro)) include hematologic and central nervous manifestations.


Subject(s)
Genetic Diseases, X-Linked/genetics , Metabolism, Inborn Errors/genetics , Mutation, Missense , Phosphoglycerate Kinase/deficiency , Black or African American , Amino Acid Substitution , Child, Preschool , Humans , Male , Phosphoglycerate Kinase/genetics
6.
Parkinsonism Relat Disord ; 64: 319-323, 2019 07.
Article in English | MEDLINE | ID: mdl-30975619

ABSTRACT

BACKGROUND: Phosphoglycerate kinase-1 deficiency is caused by X-linked recessive mutations in PGK-1 and associated with haemolytic anaemia, rhabdomyolysis, myopathy and nervous system involvement. Some cases have been rarely associated with juvenile Parkinsonism however the causal relationship between PGK1 deficiency and nigrostriatal dysfunction causing Parkinsonism has not been determined. OBJECTIVE AND METHODS: To investigate the nigrostriatal system using 99mTc-TRODAT-1 SPECT binding and report the phenotype of three affected males with early onset levodopa responsive Parkinsonism harbouring the c.491 A > T/p.D164V pathogenic variant. RESULTS: All patients initially presented with infantile-onset encephalopathic and stroke-like episodes, haemolytic anaemia and epilepsy. Two patients had an early-onset and one juvenile-onset levodopa responsive Parkinsonism with motor fluctuations. 99mTc-TRODAT-1 SPECT showed severe bilateral reduced putaminal uptake in the three patients. None of the patients had structural lesions that could explain either pre- or postsynaptic dopaminergic dysfunction. CONCLUSION: These cases provide strong evidence of a causal relationship between PGK1 deficiency and nigrostriatal pathology causing Parkinsonism. These findings have potential implications for our understanding of the pathophysiology of nigrostriatal degeneration in sporadic PD.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/metabolism , Genetic Diseases, X-Linked/metabolism , Metabolism, Inborn Errors/metabolism , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/physiopathology , Phosphoglycerate Kinase/deficiency , Putamen/metabolism , Substantia Nigra/metabolism , Adult , Age of Onset , Anemia, Hemolytic/etiology , Genetic Diseases, X-Linked/complications , Genetic Diseases, X-Linked/diagnostic imaging , Humans , Male , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/diagnostic imaging , Organotechnetium Compounds , Parkinsonian Disorders/diagnostic imaging , Parkinsonian Disorders/etiology , Pedigree , Phosphoglycerate Kinase/metabolism , Radiopharmaceuticals , Substantia Nigra/diagnostic imaging , Tomography, Emission-Computed, Single-Photon , Tropanes
7.
Biochem J ; 476(8): 1303-1321, 2019 04 30.
Article in English | MEDLINE | ID: mdl-30988012

ABSTRACT

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.


Subject(s)
Autophagosomes/immunology , Leishmania major , Macrophages , Models, Molecular , Phosphoglycerate Kinase , Protozoan Proteins , Animals , Leishmania major/genetics , Leishmania major/immunology , Leishmania major/pathogenicity , Macrophages/immunology , Macrophages/parasitology , Mice , Mice, Inbred BALB C , Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/deficiency , Phosphoglycerate Kinase/immunology , Protein Structure, Secondary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/immunology
8.
J Inherit Metab Dis ; 42(5): 803-808, 2019 09.
Article in English | MEDLINE | ID: mdl-30887539

ABSTRACT

Phosphoglycerate kinase (PGK) deficiency is a rare X-linked metabolic disorder caused by mutations in the PGK1 gene. Patients usually develop various combinations of nonspherocytic hemolytic anemia (NSHA), myopathy, and central nervous system disorders. In this national multicenter observational retrospective study, we recorded all known French patients with PGK deficiency, and 3 unrelated patients were identified. Case 1 was a 32-year-old patient with severe chronic axonal sensorimotor polyneuropathy resembling Charcot-Marie-Tooth (CMT) disease, mental retardation, microcephaly, ophthalmoplegia, pes cavus, and the new c.323G > A PGK1 hemizygous mutation. Case 2 was a 71-year-old patient with recurrent exertional rhabdomyolysis, and a c.943G > A PGK1 hemizygous mutation. Case 3 was a 48-year-old patient with NSHA, retinitis pigmentosa, mental retardation, seizures, stroke, parkinsonism, and a c.491A > T PGK1 hemizygous mutation. This study confirms that PGK deficiency is an extremely rare disorder with a wide phenotypic spectrum, and demonstrates for the first time that PGK deficiency may affect the peripheral nervous system and present as a CMT-like disorder.


Subject(s)
Genetic Diseases, X-Linked/diagnosis , Genetic Diseases, X-Linked/genetics , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Phosphoglycerate Kinase/deficiency , Adult , Aged , France , Humans , Male , Middle Aged , Mutation , Phosphoglycerate Kinase/genetics , Retrospective Studies
10.
Neurology ; 91(11): e1077-e1082, 2018 09 11.
Article in English | MEDLINE | ID: mdl-30111548

ABSTRACT

OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.


Subject(s)
Exercise Tolerance/physiology , Genetic Diseases, X-Linked/enzymology , Genetic Diseases, X-Linked/physiopathology , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/physiopathology , Phosphoglycerate Kinase/deficiency , Phosphoglycerate Kinase/metabolism , Ergometry , Exercise Test , Genetic Diseases, X-Linked/complications , Genetic Diseases, X-Linked/diagnosis , Humans , Intellectual Disability/blood , Intellectual Disability/complications , Intellectual Disability/enzymology , Intellectual Disability/physiopathology , Lactic Acid/blood , Male , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/diagnosis , Muscle, Skeletal/metabolism , Muscular Diseases/blood , Muscular Diseases/complications , Muscular Diseases/enzymology , Muscular Diseases/physiopathology , Phenotype , Phosphoglycerate Kinase/blood
12.
Brain Dev ; 40(2): 150-154, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28801086

ABSTRACT

We report the case of an 18-year-old man with a phosphoglycerate kinase (PGK) deficiency who had slowly progressive leukodystrophy during adolescence. The patient had a history of severe neonatal jaundice, hemolytic crisis with rhabdomyolysis triggered by febrile viral infections, dysarthria, and intellectual disability during early childhood. Clumsiness in walking and writing became obvious at ∼10years of age. Evaluations performed by us on the 18-year-old patient confirmed the presence of pyramidal tract signs, increased muscle tone, and generalized dystonia. Brain magnetic resonance (MR) imaging revealed leukodystrophy in the periventricular white matter, posterior limbs of the internal capsule, dorsal pons, and middle cerebellar peduncles. Compared to MR images acquired at 9years of age, MR images acquired at 18years of age showed that the white matter atrophy had progressed. The PGK deficiency was diagnosed by identifying a known missense mutation in PGK1 (c.1060G>C) through comprehensive target capture sequencing and by observing low PGK activity in his red blood cells. The patient underwent a ketogenic diet for 2weeks, which we expected would increase adenosine triphosphate levels through sources other than the PGK-associated glycolytic pathway. The diet was not tolerated owing to the unexpected emergence of hemolysis. Hemolytic anemia, neurological dysfunction, and myopathy are often associated with PGK deficiencies. However, leukodystrophy as a symptom of PGK deficiency has not been reported previously. Our case highlights the progressive nature of the neurological complications related to PGK deficiencies. Therefore, long-term follow-up is recommended, even if neurological impairments are not obvious during childhood.


Subject(s)
Genetic Diseases, X-Linked/complications , Genetic Diseases, X-Linked/physiopathology , Leukoencephalopathies/complications , Leukoencephalopathies/physiopathology , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/physiopathology , Phosphoglycerate Kinase/deficiency , Adolescent , Brain/diagnostic imaging , Diagnosis, Differential , Diet, Ketogenic/adverse effects , Disease Progression , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/therapy , Humans , Male , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/therapy , Phosphoglycerate Kinase/genetics
13.
Oncogene ; 36(27): 3868-3877, 2017 07 06.
Article in English | MEDLINE | ID: mdl-28263974

ABSTRACT

High aerobic glycolysis not only provides energy to cancer cells, but also supports their anabolic growth. JMJD1A, a histone demethylase that specifically demethylates H3K9me1/2, is overexpressed in multiple cancers, including urinary bladder cancer (UBC). It is unclear whether JMJD1A could promote cancer cell growth through enhancing glycolysis. In this study, we found that downregulation of JMJD1A decreased UBC cell proliferation, colony formation and xenograft tumor growth. Knockdown of JMJD1A inhibited glycolysis by decreasing the expression of genes participated in glucose metabolism, including GLUT1, HK2, PGK1, PGM, LDHA and MCT4. Mechanistically, JMJD1A cooperated with hypoxia inducible factor 1α (HIF1α), an important transcription factor for glucose metabolism, to induce the glycolytic gene expression. JMJD1A was recruited to the promoter of glycolytic gene PGK1 to demethylate H3K9me2. However, the JMJD1A (H1120Y) mutant, which loses the demethylase activity, failed to cooperate with HIF1α to induce the glycolytic gene expression, and failed to demethylate H3K9me2 on PGK1 promoter, suggesting that the demethylase activity of JMJD1A is essential for its coactivation function for HIF1α. Inhibition of glycolysis through knocking down HIF1α or PGK1 decelerated JMJD1A-enhanced UBC cell growth. Consistent with these results, a positive correlation between JMJD1A and several key glycolytic genes in human UBC samples was established by analyzing a microarray-based gene expression profile. In conclusion, our study demonstrates that JMJD1A promotes UBC progression by enhancing glycolysis through coactivation of HIF1α, implicating that JMJD1A is a potential molecular target for UBC treatment.


Subject(s)
Glycolysis , Jumonji Domain-Containing Histone Demethylases/physiology , Urinary Bladder Neoplasms/enzymology , Animals , Cell Line, Tumor , Cell Proliferation , Disease Progression , Gene Expression Regulation, Neoplastic , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/metabolism , Glucose/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Male , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/metabolism , Mice, Nude , Neoplasm Transplantation , Phosphoglycerate Kinase/deficiency , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Promoter Regions, Genetic , Response Elements , Transcription, Genetic , Transcriptional Activation , Urinary Bladder Neoplasms/pathology
14.
Neuromuscul Disord ; 26(3): 207-10, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26883264

ABSTRACT

We report two brothers with mild intellectual deficiency, exercise intolerance, rhabdomyolysis, seizures and no hemolysis. Phosphoglycerate kinase (PGK) activity was strongly decreased in their red blood cells. Subsequent molecular analysis of PGK1 revealed hemizygosity for a novel mutation c.756 + 3A > G, in intron 7. Analysis of the effect of this mutation on pre-mRNA processing demonstrated markedly decreased levels of normal PGK1 mRNA. In addition, the c.756 + 3A > G change resulted in abnormally spliced transcripts. If translated, these transcripts mostly encode for C-terminally truncated proteins. The consequences of the c.756 + 3A > G mutation is discussed, as well as the genotype-to-phenotype correlation with regard to previously described mutations (PGK Fukuroi and PGK Antwerp), which also result in C-terminal truncated proteins.


Subject(s)
Genetic Diseases, X-Linked/diagnosis , Genetic Diseases, X-Linked/genetics , Intellectual Disability/complications , Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Myoglobinuria/complications , Phosphoglycerate Kinase/deficiency , Seizures/complications , Adolescent , Genetic Diseases, X-Linked/complications , Genotype , Hemolysis , Humans , Male , Metabolism, Inborn Errors/complications , Muscle, Skeletal/pathology , Mutation , Phenotype , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/ultrastructure , Siblings
15.
Doc Ophthalmol ; 131(3): 215-20, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26396085

ABSTRACT

PURPOSE: Phosphoglycerate kinase (PGK) deficiency is an X-linked neurometabolic genetic disorder with variable systemic manifestations. So far, only one patient with retinal anomalies has been reported, but no visual electrophysiology findings were described. We report the first description of visual electrophysiology in a child with PGK deficiency. This provides further information for the site of involvement in the eye. METHOD: A case history of a nine-year-old boy with PGK deficiency is reported. RESULTS: This patient was diagnosed with PGK deficiency by screening soon after birth, as his mother was a known carrier of a PGK gene mutation. A bone marrow transplant was performed at the age of 9 months. He had two episodes of encephalopathy following the transplant but no acute episode of haemolysis. From the age of 6 years, his vision has been deteriorating. Visual electrophysiology results identified retinal involvement involving both rod and cone dysfunction. The visual evoked potential was normal. CONCLUSIONS: Retinal dystrophy may be one of the clinical manifestations of phosphoglycerate kinase deficiency.


Subject(s)
Genetic Diseases, X-Linked/physiopathology , Metabolism, Inborn Errors/physiopathology , Night Vision/physiology , Phosphoglycerate Kinase/deficiency , Photoreceptor Cells, Vertebrate/physiology , Retinal Dystrophies/physiopathology , Vision Disorders/physiopathology , Child , Electrophysiology , Electroretinography , Evoked Potentials, Visual/physiology , Follow-Up Studies , Genetic Diseases, X-Linked/genetics , Humans , Male , Metabolism, Inborn Errors/genetics , Pedigree , Phosphoglycerate Kinase/genetics , Point Mutation , Retinal Dystrophies/diagnosis , Tomography, Optical Coherence , Vision Disorders/diagnosis
16.
Int J Hematol ; 100(4): 393-7, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24934115

ABSTRACT

Phosphoglycerate kinase (PGK) deficiency, a rare X-linked inherited disorder, manifests as various combinations of hemolytic anemia, neurological dysfunction, and myopathy. We report a Japanese boy with PGK deficiency presenting as chronic hemolytic anemia. The diagnosis of PGK1 deficiency was made at 11 months of age on the basis of low PGK enzyme activity (36.7 IU/g Hb; normal, 264-326 IU/g Hb) and the identification through PGK1 gene sequencing of a novel missense mutation: c. 1180A>G at exon 10. The mutation, which has been designated PGK-Aoto, results in a Thr394Ala amino-acid substitution at ß-strand L. Because ß-strand L plays an important role in the function of the hinge connecting the two domains of PGK, the Thr394Ala substitution may perturb this motion. At 3 years of age the patient has transfusion-dependent hemolytic anemia but no evidence of neuromuscular disease or developmental delay. Long-term follow-up will be needed to identify possible future clinical manifestations.


Subject(s)
Anemia, Hemolytic , Exons , Genetic Diseases, X-Linked , Metabolism, Inborn Errors , Mutation, Missense , Phosphoglycerate Kinase/deficiency , Amino Acid Substitution , Anemia, Hemolytic/complications , Anemia, Hemolytic/enzymology , Anemia, Hemolytic/genetics , Asian People , Child, Preschool , Chronic Disease , Genetic Diseases, X-Linked/complications , Genetic Diseases, X-Linked/enzymology , Genetic Diseases, X-Linked/genetics , Humans , Male , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/genetics , Phosphoglycerate Kinase/genetics
17.
J Inherit Metab Dis ; 37(6): 909-16, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24838780

ABSTRACT

Mutations in genes encoding metabolic enzymes are often the cause of inherited diseases. Mutations usually affect the ability of proteins to fold properly, thus leading to enzyme loss of function. In this work, we explored the relationships between protein stability, aggregation, and degradation in vitro and inside cells in a large set of mutants associated with human phosphoglycerate kinase 1 (hPGK1) deficiency. To this end, we studied a third of the pathogenic alleles reported in the literature using expression analyses and biochemical, biophysical, and computational procedures. Our results show that most pathogenic variants studied had an increased tendency to aggregate when expressed in Escherichia coli, well correlating with the denaturation half-lives measured by thermal denaturation in vitro. Further, the most deleterious mutants show reduced stability toward chemical denaturation and proteolysis, supporting a pivotal role of thermodynamic stability in the propensity toward aggregation and proteolysis of pathogenic hPGK1 mutants in vitro and inside cells. Our strategy allowed us to unravel the complex relationships between protein stability, aggregation, and degradation in hPGK1 deficiency, which might be used to understand disease mechanisms in many inborn errors of metabolism. Our results suggest that pharmacological chaperones and protein homeostasis modulators could be considered as good candidates for therapeutic approaches for hPGK1 deficiency.


Subject(s)
Genetic Diseases, X-Linked/genetics , Metabolism, Inborn Errors/genetics , Phosphoglycerate Kinase/deficiency , Alleles , Enzyme Stability , Escherichia coli , Homeostasis , Humans , Mutation , Phosphoglycerate Kinase/genetics , Protein Conformation , Protein Denaturation , Proteolysis
18.
Biochimie ; 103: 7-15, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24721582

ABSTRACT

Human phosphoglycerate kinase 1 (hPGK1) is a glycolytic enzyme essential for ATP synthesis, and it is implicated in different pathological conditions such as inherited diseases, oncogenesis and activation of drugs for cancer and viral treatments. Particularly, mutations in hPGK1 cause human PGK1 deficiency, a rate metabolic conformational disease. We have recently found that most of these mutations cause protein kinetic destabilization by significant changes in the structure/energetics of the transition state for irreversible denaturation. In this work, we explore the relationships between protein conformation, thermodynamic and kinetic stability in hPGK1 by performing comprehensive analyses in a wide pH range (2.5-8). hPGK1 remains in a native conformation at pH 5-8, but undergoes a conformational transition to a molten globule-like state at acidic pH. Interestingly, hPGK1 kinetic stability remains essentially constant at pH 6-8, but is significantly reduced when pH is decreased from 6 to 5. We found that this decrease in kinetic stability is caused by significant changes in the energetic/structural balance of the denaturation transition state, which diverge from those found for disease-causing mutations. We also show that protein kinetic destabilization by acidic pH is strongly linked to lower thermodynamic stability, while in disease-causing mutations seems to be linked to lower unfolding cooperativity. These results highlight the plasticity of the hPGK1 denaturation mechanism that responds differently to changes in pH and in disease-causing mutations. New insight is presented into the different factors contributing to hPGK1 thermodynamic and kinetic stability and the role of denaturation mechanisms in hPGK1 deficiency.


Subject(s)
Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/metabolism , Protein Denaturation , Enzyme Stability , Genetic Diseases, X-Linked/enzymology , Humans , Hydrogen-Ion Concentration , Kinetics , Metabolism, Inborn Errors/enzymology , Phosphoglycerate Kinase/deficiency , Solvents/chemistry , Thermodynamics
19.
Biochim Biophys Acta ; 1834(12): 2502-11, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23911916

ABSTRACT

Conformational diseases often show defective protein folding efficiency in vivo upon mutation, affecting protein properties such as thermodynamic stability and folding/unfolding/misfolding kinetics as well as the interactions of the protein with the protein homeostasis network. Human phosphoglycerate kinase 1 (hPGK1) deficiency is a rare inherited disease caused by mutations in hPGK1 that lead to loss-of-function. This disease offers an excellent opportunity to explore the complex relationships between protein stability and dynamics because of the different unfolding mechanisms displayed towards chemical and thermal denaturation. This work explores these relationships using two thermostable mutants (p.E252A and p.T378P) causing hPGK1 deficiency and WT hPGK1 using proteolysis and chemical denaturation. p.T378P is degraded ~30-fold faster at low protease concentrations (here, the proteolysis step is rate-limiting) and ~3-fold faster at high protease concentrations (where unfolding kinetics is rate-limiting) than WT and p.E252A, indicating that p.T378P is thermodynamically and kinetically destabilized. Urea denaturation studies support the decrease in thermodynamic stability and folding cooperativity for p.T378P, as well as changes in folding/unfolding kinetics. The present study reveals changes in the folding landscape of hPGK1 upon mutation that may affect protein folding efficiency and stability in vivo, also suggesting that native state stabilizers and protein homeostasis modulators may help to correct folding defects in hPGK1 deficiency. Moreover, detailed kinetic proteolysis studies are shown to be powerful and simple tools to provide deep insight into mutational effects on protein folding and stability in conformational diseases.


Subject(s)
Genetic Diseases, X-Linked/enzymology , Metabolism, Inborn Errors/enzymology , Mutation, Missense , Phosphoglycerate Kinase/deficiency , Protein Denaturation , Proteolysis , Amino Acid Substitution , Enzyme Stability , Genetic Diseases, X-Linked/genetics , Humans , Metabolism, Inborn Errors/genetics , Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Urea/chemistry
20.
Biochemistry ; 52(7): 1160-70, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23336698

ABSTRACT

Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease.


Subject(s)
Genetic Diseases, X-Linked/enzymology , Metabolism, Inborn Errors/enzymology , Phosphoglycerate Kinase/chemistry , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Calorimetry, Differential Scanning , Enzyme Stability , Genetic Diseases, X-Linked/genetics , Humans , Kinetics , Metabolism, Inborn Errors/genetics , Models, Chemical , Mutation , Phosphoglycerate Kinase/deficiency , Protein Denaturation , Protein Folding , Temperature , Urea/chemistry
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