X-Linked Levodopa-Responsive Parkinsonism-Epilepsy Syndrome: A Novel PGK1 Mutation and Literature Review.

BACKGROUND: Genetic underpinnings in Parkinson's disease (PD) and parkinsonian syndromes are challenging, and recent discoveries regarding their genetic pathways have led to potential gene-specific treatment trials. CASES: We report 3 X-linked levodopa (l-dopa)-responsive parkinsonism-epilepsy syndrome cases due to a hemizygous variant in the phosphoglycerate kinase 1 (PGK1) gene. The likely pathogenic variant NM_000291.4 (PGK1):c.950G > A;p.(Gly317Asp) was identified in a hemizygous state. LITERATURE REVIEW: Only 8 previous cases have linked this phenotype to PGK1, a gene more commonly associated with hemolytic anemia and myopathy. The unusual association of epilepsy, psychiatric symptoms, action tremor, limb dystonia, cognitive symptoms, and l-dopa-responsive parkinsonism must draw attention to PGK1 mutations, especially because this gene is absent from most commercial hereditary parkinsonism panels. CONCLUSIONS: This report aims to shed light on an overlooked gene that causes hereditary parkinsonian syndromes. Further research regarding genetic pathways in PD may provide a better understanding of its pathophysiology and open possibilities for new disease-modifying trials, such as SNCA, LRRK2, PRKN, PINK1, and DJ-1 genes.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli.

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with KmATP values of 0.13 mM and 0.5 mM, and Km3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8-10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli.

A constitutive expression system for Pichia pastoris based on the PGK1 promoter.

OBJECTIVES: To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter. RESULTS: P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants. CONCLUSIONS: A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance.

The neomycin-resistance (neo(r)) gene is widely used as a selectable marker in eukaryotic expression vectors; however, its expression often affects that of target genes. Cre recombinase recognizes LoxP sites, leading to site-specific recombination and deletion of DNA and RNA between two LoxP sites. In the present study, a humanized Fat-1 gene (hFat-1) was generated by DNA Works and used to construct a pC-PGK-neo(r)-hfat-1 expression vector, in which PGK-neo(r) was flanked by two LoxP sites. The pC-PGK-neo(r)-hfat-1 plasmids were transfected into porcine fetal fibroblasts using liposomes, and three transgenic cell lines were obtained by culturing with 400 µg/mL G418 for 7 days. Next, these cell lines were transfected with a Cre recombinase expression plasmid, which contains a puromycin resistance gene, in order to delete neo(r), which was integrated into the genome. hFat-1-neo(r) negative cells were obtained following puromycin selection. Real-time quantitative polymerase chain reaction data indicated that neomycin-resistant cells had higher hFat-1 expression than neomycin-sensitive cells. High performance gas chromatography data suggested that the n-6/n-3 ratio was significantly lower in transfected cells than in wild-type cells. The n-6/n-3 ratio in Cre-treated hFat-1-transfected cells was higher than that in untreated cells, suggesting that deletion of PGK-neo(r) decreased hFat-1 expression.

Leishmania major phosphoglycerate kinase transcript and protein stability contributes to differences in isoform expression levels.

Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.

The glycosomal-membrane associated phosphoglycerate kinase isoenzyme A plays a role in sustaining the glucose flux in Trypanosoma cruzi epimastigotes.

In Trypanosoma cruzi three isoenzymes of phosphoglycerate kinase (PGK) are found which are simultaneously expressed: the cytosolic isoenzyme PGKB as well as two glycosomal enzymes, PGKA and PGKC. In this paper, we show that PGKA in T. cruzi epimastigotes is associated to the glycosomal membrane; it is responsible for about 23% of the glycosomal PGK activity, the fraction that remains in the pellet after osmotic shock treatment of purified organelles, in contrast to the 77% soluble activity that is mainly attributed to PGKC. Antibodies against the unique 80 amino-acid insertion of PGKA blocked almost completely the glucose consumption by epimastigotes that were partially permeabilized with digitonin. These results indicate that PGKA is the predominant isoenzyme for sustaining glycolysis through the glycosomes of these parasites.

The phosphoglycerate kinase isoenzymes have distinct roles in the regulation of carbohydrate metabolism in Trypanosoma cruzi.

The glycolytic enzyme phosphoglycerate kinase (PGK) is present in Trypanosoma cruzi as three isoenzymes, two of them located inside glycosomes (PGKA and PGKC) and another one in the cytosol (PGKB). The three isoenzymes are expressed at all stages of the life cycle of the parasite. A heterologous expression system for PGKA (rPGKA) was developed and the substrate affinities of the natural and recombinant PGKA isoenzyme were determined. Km values measured for 3-phosphoglycerate (3PGA) were 174 and 850 µM, and for ATP 217 and 236 µM, for the natural and recombinant enzyme, respectively. No significant differences were found between the two forms of the enzyme. The rPGKA was inhibited by Suramin with Ki values of 10.08 µM and 12.11 µM for ATP and 3PGA, respectively, and the natural enzyme was inhibited at similar values. A site-directed mutant was created in which the 80 amino acids PGKA sequence, present as a distinctive insertion in the N-terminal domain, was deleted. This internally truncated PGKA showed the same Km values and specific activity as the full-length rPGKA. The natural PGKC isoenzyme was purified from epimastigotes and separated from PGKA through molecular exclusion chromatography and its kinetic characteristics were determined. The Km value obtained for 3PGA was 192 µM, and 10 µM for ATP. Contrary to PGKA, the activity of PGKC is tightly regulated by ATP (substrate inhibition) with a Ki of 270 µM, suggesting a role for this isoenzyme in regulating metabolic fluxes inside the glycosomes.

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function.

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.

Gene expression, autonomic function and chronic hypoxia:lessons from the Andes.

Autonomic function is altered by altitude in sojourners and natives. We hypothesized that these physiologic responses are modulated by changes in gene expression. We compared gene product levels in 20 natives of Cerro de Pasco (CP), (4338 m), 10 of which had chronic mountain sickness (CMS) established by a CMS-scoring system, with gene products in the same men after 1 h at sea level. We further compared the results with those obtained from 10 US men residing at 1500 m. We measured gene products in white cells by reverse transcription polymerase chain reaction (RT-PCR). We focused on genes important in vascular autonomic physiology, and/or activated by hypoxia; hypoxia inducible factor 1-alpha (HIF 1-alpha), 2 splicing variants of vascular endothelial growth factor (VEGF); VEGF-121, VEGF-165, and phosphoglycerate kinase 1 (PGK 1). Normal CP natives showed high expression of all genes in CP, compared to US controls. Within 1 h of arrival at sea level, they had comparable levels to US residents. In CMS, the gene products were higher in CP. Although gene products decreased in Lima in this group, they never reached US values. VEGF 121 and 165 were correlated (P<0.001). VEGF 165 was higher in CMS in CP (P=0.006), and was positively correlated with CMS-score (R=0.86, P<0.001), and negatively correlated with arterial saturation (R=-0.79, P<0.001). Our findings underscore the changes in gene expression levels in intact humans in response to environmental stress. These changes may support the physiologic alterations induced by the ambient hypoxia at altitude and impact organism survival. They also suggest therapeutic strategies for autonomic and neurodegenerative diseases at sea level.

Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris.

We report the cloning of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris by a PCR approach. The coding sequence of the PGK1 gene comprises 1251 bp with the potential to encode a polypeptide of 416 amino acid residues, which shows high identity to homologous proteins from other yeasts. The promoter region of this gene (P(PGK1)) contains regulatory cis-elements found in other PGK1 genes, such as TATA box, CT-rich block and a heat shock element. In the 3' downstream region we identified a tripartite element 5'-TAG-TAGT-TTT-3', which is supposed to be important for transcription termination. As in other yeasts, the PGK1 gene from P. pastoris is present as a single-copy gene. Northern blot analysis revealed that the gene is transcribed as a 1.5 kb mRNA; when cells are grown on glucose the levels of this mRNA are increased two-fold in comparison to cells grown on glycerol. The transcriptional regulation of this gene by the carbon source was further confirmed when the alpha-amylase gene from Bacillus subtilis was placed under the control of P(PGK1): higher levels of expression were obtained when cells were grown on glucose as compared to glycerol and methanol. Preliminary results related to the strength of P(PGK1) show that it represents a potential alternative to constitutive heterologous expression in P. pastoris.

The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi.

In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane.

Organization, sequence and stage-specific expression of the phosphoglycerate kinase genes of Leishmania mexicana mexicana.

In Leishmania mexicana two genes were detected coding for different isoforms of the glycolytic enzyme phosphoglycerate kinase. This situation contrasts with that observed in other Trypanosomatidae (Trypanosoma brucei, Trypanosoma congolense, Crithidia fasciculata) analyzed previously, which all contain three different genes coding for isoenzymes A, B and C, respectively. All attempts to detect in L. mexicana a type A PGK, or a gene encoding it, proved unsuccesful. We have cloned and characterized the genes PGKB and PGKC. They code for polypeptides of 416 and 478 amino acids with a molecular mass of 45146 and 51318 Da, respectively. The two polypeptides are 99% identical. PGKC is characterized by a 62 residue C-terminal extension with alternating stretches of hydrophobic and charged, mainly positive amino acids. As in other Trypanosomatidae, PGKB is located in the cytosol, PGKC in the glycosomes. However, Leishmania mexicana distinguishes itself from other trypanosomatids by the simultaneous expression of these isoenzymes: approximately 80% of PGK activity is found in the cytosol and 20% in the glycosomes, both in promastigotes and in the amastigote-like form of the parasite.

Lack of a BglII site at the 5' region of the PGK 1 locus: a new variant discovered in two Chibchan Amerindian groups from Costa Rica.

A new 3.8-kb allele at the 5' region of the PGK 1 locus detected by the probe pSPT/PGK is reported. This variant was discovered in the Cabecar and Guaymi, two Chibchan Amerindian groups of Costa Rica. So far, a polymorphism that consists of an EcoRI/BglI (1.3-kb) variable site within an EcoRI/BglII (1.7-kb) fragment when DNA is simultaneously digested with EcoRI, BglI and BglII is known to occur in black and Caucasian populations. These two alleles were also found in the Amerindians tested. The newly described band is due to the lack of the BglII site situated 1.7 kb downstream from the EcoRI site and to the cleavage of another BglII site 2.1 kb downstream from the lacking one. This variant might be restricted to some Amerindian groups and perhaps also to Asiatic populations. Thus, it could be a useful marker in evolutive studies and for forensic applications. Moreover, the presence of a third allele in populations with Amerindian ancestry can increase the heterozygosity of the region disclosed by the pSPT/PGK probe, thus improving its application in issues dealing with X-chromosome activation ratios in females.