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Introduction
Juan Carlos Aledo currently works at the Department of Biochemistry and Molecular Biology, University of Malaga, where he carries out research in Evolutionary Biology. Their current project is 'Methionine oxidation prediction'.
Publications
Publications (92)
The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence...
The relative contribution of mutation and selection to the amino acid substitution rates observed in empirical matrices is unclear. Herein, we present a neutral continuous fitness-stability model, inspired by the Arrhenius law (qij=aije−ΔΔGij). The model postulates that the rate of amino acid substitution (i→j) is determined by the product of a pre...
The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a new pine GS1 ( PpGS1b.2 ) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the pre...
Methionine-rich prion-like proteins can regulate liquid–liquid phase separation processes in response to stresses. To date, however, very few proteins have been identified as methionine-rich prion-like. Herein, we have performed a computational survey of the human proteome to search for methionine-rich prion-like domains. We present a census of 51...
Background
Complex enzymatic models are required for analyzing kinetic data derived under conditions that may not satisfy the assumptions associated with Michaelis–Menten kinetics. To analyze these data, several software packages have been developed. However, the complexity introduced by these programs is often dispensable when analyzing data confo...
Alignment-free methods for sequence comparison and phylogeny inference have attracted a great deal of attention in recent years. Several algorithms have been implemented in diverse software packages. Despite the great number of existing methods, most of them are based on word statistics. Although they propose different filtering and weighting strat...
Alignment-free methods for sequence comparison and phylogeny inference have attracted a great deal of attention in recent years. Several algorithms have been implemented in diverse software packages. Despite the great number of existing methods, most of them are based on word statistics. Although they propose different filtering and weighting strat...
Membraneless organelles are non-stoichiometric supramolecular structures in the micron scale. These structures can be quickly assembled/disassembled in a regulated fashion in response to specific stimuli. Membraneless organelles contribute to the spatiotemporal compartmentalization of the cell, and they are involved in diverse cellular processes of...
Methionine sulfoxidation is a post-translational modification playing important roles in cell signaling. Herein, we present ptm, an R package for the study of this modification. However, since many of the analyses applied to methionine modification can be extended to other modifications, the package can be useful to thoroughly analyze post-translat...
We are living in the Big Data era, and yet we may have serious troubles when dealing with a handful of kinetic data if we are not properly instructed. The aim of this paper, related to enzyme kinetics, is to illustrate how to determine the Km and Vmax of a michaelian enzyme avoiding the pitfalls in which we often fall. To this end, we will resort t...
Methionine oxidation plays a relevant role in cell signaling. Recently, we built a database containing thousands of proteins identified as sulfoxidation targets. Using this resource, we have now developed a computational approach aimed at characterizing the oxidation of human methionyl residues. We found that proteins oxidized in both cell-free pre...
Methionine is a proteinogenic amino acid that can be post-translationally modified. It is now well established that reactive oxygen species can oxidise methionine residues within living cells. For a long time, it has been thought that such a modification represents merely an inevitable damage derived from aerobic metabolism. However, several author...
Methionine in proteins, apart from its role in the initiation of translation, is assumed to play a simple structural role in the hydrophobic core, in a similar way to other hydrophobic amino acids such as leucine, isoleucine and valine. However, research from a number of laboratories supports the concept that methionine serves as an important cellu...
Motivation
The oxidation of protein-bound methionine to form methionine sulfoxide has traditionally been regarded as an oxidative damage. However, growing evidences support the view of this reversible reaction also as a regulatory post-translational modification. Thus, the oxidation of methionine residues has been reported to have multiple and vari...
The ability to make decisions at the cellular level is absolutely critical for the survival of organisms. Eukaryotic cells are constantly making binary decisions in response to internal and environmental signals. Among the most notable transducers of information are protein kinases. The regulation of these signaling proteins often relies on the act...
Background
The oxidation of protein-bound methionine to form methionine sulfoxide, has traditionally been regarded as an oxidative damage. However, recent evidences support the view of this reversible reaction as a regulatory post-translational modification. The perception that methionine sulfoxidation may provide a mechanism to the redox regulatio...
Background
The sulfoxidation of methionine residues within the phosphorylation motif of protein kinase substrates, may provide a mechanism to couple oxidative signals to changes in protein phosphorylation. Herein, we hypothesize that if the residues within a pair of phosphorylatable-sulfoxidable sites are functionally linked, then they might have b...
Although reactive oxygen species are best known as damaging agents linked to aerobic metabolism, it is now clear that they can also function as messengers in cellular signalling processes. Methionine, one of the two sulphur containing amino acids in proteins, is liable to be oxidized by a well-known reactive oxygen species: hydrogen peroxide. The a...
Protein phosphorylation is one of the most prevalent and well-understood protein modifications. Oxidation of protein-bound methionine, which has been traditionally perceived as an inevitable damage derived from oxidative stress, is now emerging as another modification capable of regulating protein activity during stress conditions. However, the mec...
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 methi...
In contraposition to the view of aging as a stochastic time-dependent accumulation of damage, phenoptotic theories of aging postulate that senescence may provide supra-individual advantages, and therefore it might have been promoted by natural selection. We reason that although programmed aging theories are subjectively appealing because they conve...
Respiratory complexes are encoded by two genomes (mitochondrial DNA [mtDNA] and nuclear DNA [nDNA]). Although the importance of intergenomic coadaptation is acknowledged, the forces and constraints shaping such coevolution are largely unknown. Previous works using cytochrome c oxidase (COX) as a model enzyme have led to the so-called “optimizing in...
The quest to understand why and how we age has led to numerous lines of investigation that have gradually converged to consider mitochondrial metabolism as a major player. During mitochondrial respiration a small and variable amount of the consumed oxygen is converted to reactive species of oxygen (ROS). For many years, these ROS have been perceive...
Brain volume and cheek-tooth size have traditionally been considered as two traits that show opposite evolutionary trends during the evolution of Homo. As a result, differences in encephalization and molarization among hominins tend to be interpreted in paleobiological grounds, because both traits were presumably linked to the dietary quality of ex...
Membrane lipid composition is an important correlate of the rate of aging of animals and, therefore, the determination of their longevity. In the present work, the use of high-throughput technologies allowed us to determine the plasma lipidomic profile of 11 mammalian species ranging in maximum longevity from 3.5 to 120 years. The non-targeted appr...
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...
By using a combination of evolutionary and structural data from 231 species, we have addressed the relationship between evolution and structural features of cytochrome b and COX I, two mtDNA-encoded proteins. The interior of cytochrome b, in contrast to that of COX I, exhibits a remarkable tolerance to changes. The higher evolvability of cytochrome...
This is a response to Sidney Toby’s letter (DOI: 10.1021/ed100288j) about the article “The Effect of Temperature on the Enzyme-Catalyzed Reaction: Insights from Thermodynamics” (DOI: 10.1021/ed800088n).
We have investigated the effect of chronic competitive inhibition of SNAT2 (System A) amino acid (AA) transport, induced by incubation with a saturating dose of a non-metabolisable System A amino acid analogue (Me-AIB), on growth and proliferation of MCF-7 human breast cancer cells in complete culture medium. These cells express Na+- and pH-depende...
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 s...
When teaching the effect of temperature on biochemical reactions, the problem is usually oversimplified by confining the thermal effect to the catalytic constant, which is identified with the rate constant of the elementary limiting step. Therefore, only positive values for activation energies and values greater than 1 for temperature coefficients...
Glucokinase is essential for glucose-stimulated insulin release from the pancreatic β-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 hyperinsulinis...
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 hyperinsuli...
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 hyperinsuli...
Glucokinase (GK) is essential for glucose-stimulated insulin release from pancreatic β-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of “Glucokinase Disease”, i.e. Monogenic Diabetes of Youth (GCK-MDY), Permanent Neonatal Diabetes (inactivating mutations) and Congenital Hyperi...
Energy dissipation fulfils an important function in metabolism driving the flux of matter through metabolic pathways. The flux rate is a multivariable function. One of such variables affecting the flux is the actual free energy change. A particular case of metabolic pathway is analysed to illustrate this fact, using only general enzymic and thermod...
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 a...
Glycolysis, an ancient energy-processing pathway, can operate either under an efficient but slow regime or, alternatively,
under a dissipative but fast-working regime. Trading an increase in efficiency for a decrease in rate represents a cooperative
behavior, while a dissipative metabolism can be regarded as a cheating strategy. Herein, using irrev...
A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase-interacting protein (GIP), because it interacts with the C-terminal region of the human L-type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unkno...
A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase-interacting protein (GIP), because it interacts with the C-terminal region of the human L-type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unkno...
Glutamatergic signalling plays an important role in the coordination of hormone secretion from the endocrine pancreas. Thus, glutamate production must be a process exquisitely regulated to ensure a proper transmitter function. Recently we have reported that the endocrine pancreas co-expresses two isoforms of the protein glutaminase (GA), denoted as...
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 al...
A recent paper published in PLoS Biology [1] deals with the contention of whether the rates of morphological evolution are accelerated on islands relative to the mainland. Because of the scarcity of empirical data, the long-held supposition that insular mammals can evolve faster than their continental counterparts remains debatable. In this context...
A hallmark of living matter is its ability to extract and transform energy from the environment. Not surprisingly, biology students are required to take thermodynamics. The necessity of coupling exergonic reactions to endergonic processes is easily grasped by most undergraduate students. However, when addressing the thermodynamic concept of coupled...
It has been proposed that the glycolytic stoichiometry of 2 ATP per glucose is the result of an optimization that maximizes the rate of ATP production. However, using a nonequilibrium thermodynamic approach, we show here that glycolysis operates under optimal output power and not at optimal flow of ATP production. Furthermore, it can be proved that...
The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox. Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extrac...
Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in beta-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary c...
Tumours, and in general rapidly dividing cells, behave as dissipative devices that apparently waste glutamine, since its consumption seems to exceed both energetic and nitrogen needs. Although not conclusive, there is compelling evidence suggesting that the consumption of such large amounts of glutamine is essential to sustain high rates of cellula...
Despite the utility that energy diagrams have as a teaching and learning tool, a survey of their use, in seven popular Biochemistry textbooks, reveals that there is certain confusion around this topic. In our opinion, this confusion arises from the reluctance of authors to consider and indicate the conditions under which the reaction being represen...
The first complete sequence of human L-glutaminase was deduced from breast cancer glutaminase cDNA cloned in our laboratory. This cDNA clone has now been engineered to synthesize both precursor and mature forms of the protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector...
In mammals, there are two different genes encoding for glutaminase isoforms, named liver (LGA) and kidney (KGA) types. LGA has long been believed to be present only in liver mitochondria from adult animals. However, we have recently reported the presence of LGA mRNA in human brain. We now describe the expression of LGA mRNA in the brain of other ma...
Staying alive is an energetically expensive task. Therefore, the necessity of coupling the exergonic nutrient transformation reactions to the endergonic synthesis of ATP is easily grasped by most undergraduate students. On the other hand, it is often difficult for students to understand the importance of energy dissipation. The purpose of this pape...
The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two-hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, alpha1-syntrophin and a glutaminase-interacting protein, named GIP, that showed associ...
A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids has been previously isolated in our laboratory. The protein was termed GIP (glutaminase-interacting protein) because it interacts with the C-terminal region of the human brain glutaminase L. Here we report the heterologous expression of GIP as a histidine-tagged fusio...
On Mar 18, 2003 this sequence version replaced gi:27436084.
The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two-hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, alpha1-syntrophin and a glutaminase-interacting protein, named GIP, that showed associ...
Glutaminolysis is initiated by either of two isoforms, K- and L-types, of the enzyme phosphate-activated glutaminase. The
chromosomal localization, genomic organization, and the tissue-specific expression of the genes have been investigated in
the human by using isoform-specific cDNA probes. Results obtained from radiation hybrid mapping experiment...
Phosphate-activated glutaminase has a critical role in tumours and rapidly dividing cells and its activity is correlated with malignancy. Ehrlich ascites tumour cells transfected with the pcDNA3 vector containing an antisense segment (0.28 kb) of rat kidney glutaminase showed impairment in the growth rate and plating efficiency, as well as a shorta...
Changes in the relative mRNA levels of phosphate-activated glutaminase (PAG) and glutamine synthetase (GS) in the liver and kidney of mice bearing a highly malignant strain of Ehrlich ascites tumor cells were determined at different days after tumor transplantation. Kidney glutaminase mRNA steadily increased, reaching maximum values at day 10 of tu...
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...
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...
Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambdagt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE')...
The influence of tumor implantation on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and stability was determined in the spleen of tumor-bearing mice. While GAPDH mRNA levels were not altered in skeletal muscle, kidney and liver from tumor-bearing mice, tumor implantation led to a 5.6-fold increase in the levels of splenic GAPDH mRNA...
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 modifi...
The influence of progressive tumor growth on phosphate-activated glutaminase (PAG) expression in splenocytes from mice bearing Ehrlich ascites carcinoma cells was investigated. Implantation of Ehrlich ascites tumor cells in the peritoneal cavity of mice led to a 2.3-fold stimulation of spleen PAG activity 48 h later. Four days after tumor implantat...
In skeletal muscle, acute insulin treatment results in the recruitment of the GLUT4 glucose transporter from intracellular vesicular structures to the plasma membrane. The precise nature of these intracellular GLUT4 stores has, however, remained poorly defined. Using an established skeletal-muscle fractionation procedure we present evidence for the...
Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood...
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 im...
We have investigated the subcellular distribution and association of cellubrevin, a low molecular weight protein implicated in the process of membrane fusion, with intracellular membranes containing the insulin-sensitive GLUT4 glucose transporter from rat adipocytes, rat skeletal muscle and human skeletal muscle. SDS-PAGE and immunoblot analyses of...
In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the α2 subunit of the Na,K-ATPase from an intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous sucrose gradient have indicated that the insulin-induced loss of both protein...
The role of Rab4, a small molecular weight GTP binding protein implicated in endosomal/plasma membrane (PM) recycling, in the translocation of the GLUT4 transporter in rat skeletal muscle was studied. Muscle membranes, prepared by subcellular fractionation of control and insulin treated rat skeletal muscle, were subjected to SDS/PAGE and immunoblot...
The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expres...
Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of th...
Two alternative purification schemes to obtain the glutaminase from Ehrlich tumor cells in a highly purified form have been developed. One experimental approach is based on conventional and high-performance liquid chromatography fractionation techniques, yielding a 37-fold higher purification than has been previously reported. The method comprises:...
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 act...
A new procedure for the analysis and detection of phosphate-activated glutaminase (EC 3.5.1.2) by native electrophoresis has been developed. The method is based on the in situ detection of glutaminase activity in two different systems of native polyacrylamide gradient gels, containing 3-(3-cholamidopropyl)-dimethyl-ammonio-1-propane sulfonate (CHAP...