Juan Carlos Aledo

Phosphate-activated glutaminase expression during tumor development

Changes in phosphate‐activated glutaminase activities determined in intact cells and isolated mitochondria have been followed during mouse Ehrlich ascites carcinoma development. Glutaminase activities parallel the levels of poly(A)+ RNAs encoding for the mitochondrial phosphate activated glutaminase. During the exponential growth phase, maximum activity was observed and the relative abundance of glutaminase mRNA significantly increased with regard to the stationary growth phase. The presented results show that tumor phosphate‐activated glutaminase is subject to long‐term regulation by differential gene expression.

Mitochondrially encoded methionine is inversely related to longevity in mammals

Methionine residues in proteins react readily with reactive oxygen species making them particularly sensitive to oxidation. However, because oxidized methionine can be reduced back in a catalyzed reaction, it has been suggested that methionine residues act as oxidant scavengers, protecting not only the proteins where they are located but also the surrounding macromolecules. To investigate whether methionine residues may be selected for or against animal longevity, we carried out a meta‐examination of mitochondrial genomes from mammalian species. Our analyses unveiled a hitherto unnoticed observation: mitochondrially encoded polypeptides from short‐lived species are enriched in methionine when compared with their long‐lived counterparts. We show evidence suggesting that methionine addition to proteins in short‐lived species, rather than methionine loss from proteins in long‐lived species, is behind the reported difference in methionine usage. The inverse association between longevity and methionine, which persisted after correction for body mass and phylogenetic interdependence, was paralleled by the methionine codon AUA, but not by the codon AUG. Although nuclear encoded mitochondrial polypeptides exhibited higher methionine usage than nonmitochondrial proteins, correlation with longevity was only found within the group of those polypeptides located in the inner mitochondrial membrane. Based on these results, we propose that short‐lived animals subjected to higher oxidative stress selectively accumulate methionine in their mitochondrially encoded proteins, which supports the role of oxidative damage in aging.

Opposite clinical phenotypes of glucokinase disease: Description of a novel activating mutation and contiguous inactivating mutations in human glucokinase (GCK) gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.

Interrelationship between oxidative damage and antioxidant enzyme activities: An easy and rapid experimental approach

This article describes a method for determining some antioxidant enzyme activities (catalase and/or glutathione peroxidase) and the oxidative status (protein oxidative damage and/or lipid peroxidation) of human blood. However, the main objective of the work is to illustrate the relationship between antioxidant defences and oxidative damage, showing to students their correlation and the general importance of the biochemical regulation in health and diseases.

Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation.

Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants.

Switching Between Cooperation and Competition in the Use of Extracellular Glucose

This paper addresses some questions related to the evolution of cooperative behaviors, in the context of energetic metabolism. Glycolysis can perform either under a dissipative working regime suitable for rapid proliferation or under an efficient regime that entails a good modus operandi under conditions of glucose shortage. A cellular mechanism allowing switching between these two regimes may represent an evolutionary achievement. Thus, we have explored the conditions that might have favored the emergence of such an accommodative mechanism. Because of an inevitable conflict for resources between individual interests and the common good, rapid and inefficient use of glucose is always favored by natural selection in spatially homogeneous environment, regardless of the external conditions. In contrast, when the space is structured, the behavior of the system is determined by its free energy content. If the fuel is abundant, the dissipative strategy dominates the space. However, under famine conditions the efficient regime represents an evolutionary stable strategy in a Harmony game. Between these two extreme situations, both metabolic regimes are engaged in a Prisoner’s Dilemma game, where the output depends on the extracellular free energy. The energy transition values that lead from one domain to another have been calculated. We conclude that an accommodative mechanism permitting alternation between dissipative and efficient regimes might have evolved in heterogeneous and highly fluctuating environments. Overall, the current work shows how evolutionary optimization and game-theoretical approaches can be complementary in providing useful insights into biochemical systems.

The role of metabolic memory in the ATP paradox and energy homeostasis.

In yeast, a sudden transition from glucose limitation to glucose excess leads to a new steady state at increased metabolic fluxes with a sustained decrease in the ATP concentration. Although this behaviour has been rationalized as an adaptive metabolic strategy, the mechanism behind it remains unclear. Nevertheless, it is thought that, on glucose addition, a metabolite derived from glycolysis may up‐regulate ATP‐consuming reactions. The adenine nucleotides themselves have been ruled out as the signals that mediate this regulation. This is mainly because, in that case, it would be expected that the new steady state at increased fluxes would be accompanied by an increased stationary ATP concentration. In this study, we present a core model consisting of a monocyclic interconvertible enzyme system. Using a supply–demand approach, we demonstrate that this system can account for the empirical observations without involving metabolites other than the adenine nucleotides as effectors. Moreover, memory is an emerging property of such a system, which may allow the cell to sense both the current energy status and the direction of the changes.

INVOLVEMENT OF ESSENTIAL CYSTEINE AND HISTIDINE RESIDUES IN THE ACTIVITY OF ISOLATED GLUTAMINASE FROM TUMOUR CELLS

The pH dependence of the phosphate-activated glutaminase isolated from Ehrlich tumour cells suggests a functional role for two prototropic groups with apparent pKa of 9.3 and 7.7 at the active site of the protein; these pKa values are compatible with cysteine and histidine residues, respectively. This possibility was investigated by chemical modification studies of the purified enzyme. N-Ethylmaleimide fully inactivated the purified glutaminase; the reaction order was very close to 1.0, suggesting that N-ethylmaleimide modifies glutaminase at a single essential site. Spectrophotometric studies of the isolated protein treated with diethyl pyrocarbonate indicate that two histidine residues are modified. Since glutaminase is loosely associated to the inner mitochondrial membrane, modification experiments were also carried out using mitochondrial membrane fractions. N-Ethylmaleimide and diethyl pyrocarbonate gave similar results in mitochondria membrane-bound enzyme to those obtained with purified enzyme. Glutamate, which behaves as a competitive inhibitor of the enzyme, partially protected the inactivation caused by N-ethylmaleimide in membrane-bound experiments. The results suggest the existence of a critical histidine residue(s) in the tumour glutaminase, and strongly support the notion that a cysteine residue, which is located at (or near) the active site, is involved in the catalytic mechanism as well.

Mutational bias plays an important role in shaping longevity-related amino acid content in mammalian mtDNA-encoded proteins.

During the course of evolution, amino acid shifts might have resulted in mitochondrial proteomes better endowed to resist oxidative stress. However, owing to the problem of distinguishing between functional constraints/adaptations in protein sequences and mutation-driven biases in the composition of these sequences, the adaptive value of such amino acid shifts remains under discussion. We have analyzed the coding sequences of mtDNA from 173 mammalian species, dissecting the effect of nucleotide composition on amino acid usages. We found remarkable cysteine avoidance in mtDNA-encoded proteins. However, no effect of longevity on cysteine content could be detected. On the other hand, nucleotide compositional shifts fully accounted for threonine usages. In spite of a strong effect of mutational bias on methionine abundances, our results suggest a role of selection in determining the composition of methionine. Whether this selective effect is linked or not to protection against oxidative stress is still a subject of debate.