Héctor Quezada

Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma

It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.

Saccharomyces cerevisiae Bat1 and Bat2 Aminotransferases Have Functionally Diverged from the Ancestral-Like Kluyveromyces lactis Orthologous Enzyme

Background Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. Principal Findings Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. Conclusions Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism.

Specialization of the paralogue LYS21 determines lysine biosynthesis under respiratory metabolism in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the first committed step of the lysine biosynthetic pathway is catalysed by two homocitrate synthases encoded by LYS20 and LYS21. We undertook a study of the duplicate homocitrate synthases to analyse whether their retention and presumable specialization have affected the efficiency of lysine biosynthesis in yeast. Our results show that during growth on ethanol, homocitrate is mainly synthesized through Lys21p, while under fermentative metabolism, Lys20p and Lys21p play redundant roles. Furthermore, results presented in this paper indicate that, in contrast to that which had been found for Lys20p, lysine is a strong allosteric inhibitor of Lys21p (K(i) 0.053 mM), which, in addition, induces positive co-operativity for alpha-ketoglutarate (alpha-KG) binding. Differential lysine inhibition and modulation by alpha-KG of the two isozymes, and the regulation of the intracellular amount of the two isoforms, give rise to an exquisite regulatory system, which balances the rate at which alpha-KG is diverted to lysine biosynthesis or to other metabolic pathways. It can thus be concluded that retention and further biochemical specialization of the LYS20- and LYS21-encoded enzymes with partially overlapping roles contributed to the acquisition of facultative metabolism.

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells.

Kinetic analysis of PFK‐1 from rodent AS‐30D, and human HeLa and MCF‐7 carcinomas revealed sigmoidal [fructose 6‐phosphate, Fru6P]‐rate curves with different Vm values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH  4+ , or K+. The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non‐essential hyperbolic activation. PFK‐1 from rat liver and heart also exhibited the mixed cooperative‐hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased Vm and increased content of inactive (T) enzyme forms. High K+ prompted increased (Fru6P) or decreased (activators) affinities; increased Vm; and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK‐1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF‐7 cells, of L > M in AS‐30D cells, and of C in HeLa cells. PFK‐1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK‐1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK‐1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK‐1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux‐control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect. J. Cell. Biochem. 113: 1692–1703, 2012.

Paralogous ALT1 and ALT2 retention and diversification have generated catalytically active and inactive aminotransferases in Saccharomyces cerevisiae.

Background Gene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase. Principal Findings Our results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function. Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae

ABSTRACT Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine–α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

The Lys20 homocitrate synthase isoform exerts most of the flux control over the lysine synthesis pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the first committed step in the lysine (Lys) biosynthetic pathway is catalysed by the Lys20 and Lys21 homocitrate synthase (HCS) isoforms. Overexpression of Lys20 resulted in eightfold increased Lys, as well as 2‐oxoglutarate pools, which were not attained by overexpressing Lys21 or other pathway enzymes (Lys1, Lys9 or Lys12). A metabolic control analysis‐based strategy, by gradually and individually manipulating the Lys20 and Lys21 activities demonstrated that the cooperative and strongly feedback‐inhibited Lys21 isoform exerted low control of the pathway flux whereas most of the control resided on the non‐cooperative and weakly feedback‐inhibited Lys20 isoform. Therefore, the higher control of Lys20 over the Lys flux represents an exception to the dogma of higher pathway control by the strongest feedback‐inhibited enzyme and points out to multi‐site engineering (HCS isoforms and supply of precursors) to increase Lys synthesis.

Molecular basis of the unusual catalytic preference for GDP/GTP in Entamoeba histolytica 3‐phosphoglycerate kinase

Phosphoglycerate kinase (EC 2.7.2.3) catalyzes reversible phosphoryl transfer from 1,3‐bisphosphoglycerate to ADP to synthesize 3‐phosphoglycerate and ATP during glycolysis. Phosphoglycerate kinases from several sources can use GDP/GTP as alternative substrates to ADP/ATP; however, the maximal velocities (Vm) reached with the guanine nucleotides are ∼ 50% of those displayed with the adenine nucleotides. By contrast, Entamoeba histolytica phosphoglycerate kinase (EC 2.7.2.10) is the only reported phosphoglycerate kinase displaying higher activity with GDP/GTP and lower affinities for the adenine nucleotides. To elucidate the molecular basis of the Entamoeba histolytica phosphoglycerate kinase selectivity for GDP/GTP, a conformational analysis was carried out on a homology model based on crystallographic structures of yeast and pig phosphoglycerate kinases. Some amino acid residues involved in the purine ring binding site not previously described were detected. Accordingly, Y239, E309 and V311 were replaced by site‐directed mutagenesis in the Entamoeba histolytica phosphoglycerate kinase gene for the corresponding amino acid residues present in the adenine nucleotide‐dependent phosphoglycerate kinases and the recombinant proteins were purified. Kinetic analysis of the enzymes showed that the single mutants Y239F, E309Q, E309M and V311L increased their catalytic efficiencies (Vm/Km) with ADP/ATP as a result of both, increased Vm and decreased Km values. Furthermore, a higher catalytic efficiency in the double mutant Y239F/E309M was achieved, which was mainly due to an increased affinity for ADP/ATP with a concomitant diminished affinity for GDP/GTP. The main Entamoeba histolytica phosphoglycerate kinase amino acid residues involved in the selectivity for guanine nucleotides were thus identified.

The 2‐oxoglutarate supply exerts significant control on the lysine synthesis flux in Saccharomyces cerevisiae

To determine the extent to which the supply of the precursor 2‐oxoglutarate (2‐OG) controls the synthesis of lysine in Saccharomyces cerevisiae growing exponentially in high glucose, top‐down elasticity analysis was used. Three groups of reactions linked by 2‐OG were defined. The 2‐OG supply group comprised all metabolic steps leading to its formation, and the two 2‐OG consumer groups comprised the enzymes and transporters involved in 2‐OG transformation into lysine and glutamate and their further utilization for protein synthesis and storage. Various 2‐OG steady‐state concentrations that produced different fluxes to lysine and glutamate were attained using yeast mutants with increasing activities of Krebs cycle enzymes and decreased activities of Lys synthesis enzymes. The elasticity coefficients of the three enzyme groups were determined from the dependence of the amino acid fluxes on the 2‐OG concentration. The respective degrees of control on the flux towards lysine (flux control coefficients) were determined from their elasticities, and were 1.1, 0.41 and −0.52 for the 2‐OG producer group and the Lys and Glu branches, respectively. Thus, the predominant control exerted by the 2‐OG supply on the rate of lysine synthesis suggests that over‐expression of 2‐OG producer enzymes may be a highly effective strategy to enhance Lys production.

Diversification of the kinetic properties of yeast NADP‐glutamate‐dehydrogenase isozymes proceeds independently of their evolutionary origin

In the yeast Saccharomyces cerevisiae, the ScGDH1 and ScGDH3 encoded glutamate dehydrogenases (NADP‐GDHs) catalyze the synthesis of glutamate from ammonium and α‐ketoglutarate (α‐KG). Previous kinetic characterization showed that these enzymes displayed different allosteric properties and respectively high or low rate of α‐KG utilization. Accordingly, the coordinated action of ScGdh1 and ScGdh3, regulated balanced α‐KG utilization for glutamate biosynthesis under either fermentative or respiratory conditions, safeguarding energy provision. Here, we have addressed the question of whether there is a correlation between the regulation and kinetic properties of the NADP‐GDH isozymes present in S. cerevisiae (ScGdh1 and ScGdh3), Kluyveromyces lactis (KlGdh1), and Lachancea kluyveri (LkGdh1) and their evolutionary history. Our results show that the kinetic properties of K. lactis and L. kluyveri single NADP‐GDHs are respectively similar to either ScGDH3 or ScGDH1, which arose from the whole genome duplication event of the S. cerevisiae lineage, although, KlGDH1 and LkGDH1 originated from a GDH clade, through an ancient interspecies hybridization event that preceded the divergence between the Saccharomyces clade and the one containing the genera Kluyveromyces, Lachancea, and Eremothecium. Thus, the kinetic properties which determine the NADP‐GDHs capacity to utilize α‐KG and synthesize glutamate do not correlate with their evolutionary origin.