Cloning, sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme.

Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5'-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physiochemical and catalytic properties.

Modulation of drug cytotoxicity by reintroduction of wild-type p53 gene (Ad5CMV-p53) in human pancreatic cancer.

Chemotherapy does not significantly improve prognosis in pancreatic cancer. New therapeutical approaches involving p53 gene replacement appear to be very encouraging due to the key role of p53 in the cell response to DNA damage. Here, we have evaluated the effectiveness of combining wild-type p53 (wt-p53) gene reintroduction (Ad5CMV-p53) and exposure to two genotoxic drugs, gemcitabine and cisplatin, in several human pancreatic cell lines. The efficiency of the combinations was clearly dependent upon timing, as assessed by cell survival determinations. Although wt-p53 transduction before drug treatment induced chemoresistance, p53 transduction in cells treated previously with gemcitabine increased cytotoxicity. Cell cycle profiles showed significant decreases in the percentage of cells in the S phase as a consequence of arrests provoked by the expression of exogenous p53, reducing the number of cells susceptible to the drug. The sensitivity of cells to cisplatin, which has a lower degree of S-phase specificity, was not modified as much by p53 gene replacement. In contrast, the recognition of the previous drug-induced DNA damage by the newly expressed wt-p53 elicited increases in sub-G1 populations, consistent with the annexin determinations and bax/bcl-2 ratios observed. Experiments on subcutaneous pancreatic xenografts corroborated the effectiveness of this approach in vivo. Thus, the combination of p53 transduction and chemotherapy, under a correct schedule of administration, appears to be a very promising therapy for human pancreatic cancer.

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16(INK4a/CDKN2), a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt-p16 cDNA (Ad5RSV-p16). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment.

A presenilin 1 mutation (Ser169Pro) associated with early-onset AD and myoclonic seizures.

OBJECTIVE: To characterize the mutation responsible for early-onset AD in a large Spanish kindred. BACKGROUND: Mutations in the presenilin 1 (PS1) gene have been identified and are known to be responsible for 18 to 50% of familial early-onset AD cases. METHODS: Patients were characterized clinically. The proband was further studied with EEG, CSF analysis, CT, brain biopsy, and histology. Other members were studied using EEG, CT, MRI, and SPECT. Genetic analysis of PS1 was performed using PCR amplification of PS1 exons and direct sequencing followed by PS1 modeling of the normal and mutant PS1 proteins. RESULTS: A novel mutation (Ser169Pro) in exon 6 of the PS1 gene was identified in different affected members. The Ser169Pro mutation is located at a site of the PS1 protein that is not a cluster of mutations. The mutation was not present in 100 general population controls and in 50 unrelated sporadic AD cases. The Ser169Pro mutation is associated with generalized myoclonic seizures several years after the initial symptoms of AD, a very early AD onset (< or =35 years), and a rapidly progressive cognitive decline. CONCLUSIONS: The absence of the PS1 Ser169Pro mutation in the general population and in sporadic AD cases together with its detection in the affected members of this kindred suggests that it is a pathogenic mutation. The serine to proline change predicts a kink in the alpha-helix of the transmembrane domain of the PS1 protein that could radically disrupt its normal structure. Further characterization of the effect of this mutation could help identify the function of the PS1 protein and the pathogenic mechanisms of AD.

A rapid procedure for eliminating chromatofocusing buffer and concentrating minor active subforms of mitochondrial malate dehydrogenase.

Mitochondrial malate dehydrogenase from several sources contains different molecular forms whose origin is still under discussion. Separation of these subforms has been achieved by chromatofocusing. A simple and rapid method, based on 5' AMP Sepharose chromatography, has been developed to concentrate mitochondrial malate dehydrogenase subforms and simultaneously remove chromatofocusing buffer.

Coarse-grained representation of protein flexibility. Foundations, successes, and shortcomings.

Flexibility is the key magnitude to understand the variety of functions of proteins. Unfortunately, its experimental study is quite difficult, and in fact, most experimental procedures are designed to reduce flexibility and allow a better definition of the structure. Theoretical approaches have become then the alternative but face serious timescale problems, since many biologically relevant deformation movements happen in a timescale that is far beyond the possibility of current atomistic models. In this complex scenario, coarse-grained simulation methods have emerged as a powerful and inexpensive alternative. Along this chapter, we will review these coarse-grained methods, and explain their physical foundations and their range of applicability.

Factors affecting L-malate activation of mitochondrial malate dehydrogenase from chicken liver.

Mitochondrial malate dehydrogenase is activated by high concentrations of L-malate. In this paper, several factors affecting this activation on chicken liver enzyme have been investigated. The results obtained show clearly that this phenomenon is an intrinsic property of this enzyme since it does not depend on pH or ionic strength of the reaction medium. However, L-malate activation decreases when NAD+ concentration diminishes (5mM----0.2 mM) in such a way that when NAD+ concentration is 0.2 mM, L-malate does not activate mitochondrial malate dehydrogenase. On the other hand, several activators of this enzymatic system, such as citrate or phosphate, also produce the elimination of this activation by L-malate; in this case, the phenomenon seems be due to a competitive binding to a regulatory site of the different metabolites implied.

alpha-Thalassaemia due to a single codon deletion in the alpha1-globin gene. Computational structural analysis of the new alpha-chain variant. Mutations in brief no. 132. Online.

A new unstable alpha-globin chain associated with alpha-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the alpha-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the alpha-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 and DK61 results in a structurally abnormal alpha-globin chain, not previously described, named Hb Clinic, which leads to the alpha-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this alpha-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer.

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

Theoretical study of the mechanisms of substrate recognition by catalase.

A variety of theoretical methods including classical molecular interaction potentials, classical molecular dynamics, and activated molecular dynamics have been used to analyze the substrate recognition mechanisms of peroxisomal catalase from Saccharomyces cerevisiae. Special attention is paid to the existence of channels connecting the heme group with the exterior of the protein. On the basis of these calculations a rationale is given for the unique catalytic properties of this enzyme, as well as for the change in enzyme efficiency related to key mutations. According to our calculations the water is expected to be a competitive inhibitor of the enzyme, blocking the access of hydrogen peroxide to the active site. The main channel is the preferred route for substrate access to the enzyme and shows a cooperative binding to hydrogen peroxide. However, the overall affinity of the main channel for H(2)O(2) is only slightly larger than that for H(2)O. Alternative channels connecting the heme group with the monomer interface and the NADP(H) binding site are detected. These secondary channels might be important for product release.

Comparative analysis of the reduction of oxaloacetate by human hepatoma and normal liver extracts.

An apparent activation of the malate dehydrogenase activity is observed in the double-reciprocal plot at high oxaloacetate concentrations when human hepatoma extracts are analyzed. This phenomenon does not occur in healthy liver samples. In hepatoma extracts, the ratio of lactate dehydrogenase to malate dehydrogenase activities becomes five-fold higher than that of normal liver. Experiments performed with mixtures of both purified enzymes and, conversely, by using oxamate, a specific inhibitor of lactate dehydrogenase, reveal that the deviation in Michaelis-Menten behavior observed is due to the oxaloacetate reductase activity of lactate dehydrogenase instead of the presence of a novel malate dehydrogenase isoenzyme.

Purification of malate dehydrogenase from chicken liver mitochondria. Existence of a small quantity of cytosolic isoenzyme.

1. A new purification method for chicken liver mitochondrial malate dehydrogenase is described. The application of affinity chromatography through 5'AMP-Sepharose and Blue-Sepharose permits to obtain homogeneous preparations, with good yields (47%), in a short time (48 hr). 2. The 5'AMP-Sepharose chromatography reveals the presence of two malate dehydrogenase species in the mitochondrial extracts. 3. A comparative study of these forms point out the cytosolic nature of the minority form and suggests that its presence could be due to a slight interaction of the cytosolic malate dehydrogenase with mitochondrial membranes.

Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate.

Mitochondrial malate dehydrogenase shows a complex regulation pattern in the presence of citrate. Previously published results indicate that this enzyme is activated by citrate in the NAD(+)----NADH direction and inhibited in the opposite direction. Moreover, high concentrations of L-malate or oxaloacetate produce deviations from the Michaelis-Menten behaviour. Results reported in this paper clearly show that citrate both activates and inhibits mitochondrial malate dehydrogenase in the same direction (NAD(+)----NADH), and in the same reaction medium, depending on substrate concentration. This surprising effect has made it necessary to propose a new kinetic mechanism that extends those previously suggested and allows us to explain both the citrate effect (activating or inhibitory) and the effect of high concentrations of L-malate and oxaloacetate.

Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Replacement of terminal cysteine with histidine in the metallothionein alpha and beta domains maintains its binding capacity.

To generate novel forms of metal-binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli. The spectroscopic and analytical results showed that the alphaMT (C33H, C36H, C41H, C57H) and betaMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected. In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd-mutated MT forms with 4 and 3 Cd(II), respectively. However, although Cys-to-His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild-type proteins. While C13H, C36H and C57H seem to contain Zn(II)-aggregates that are closely related to those of the wild-type proteins, only C41H and C57H gave rise to Cd(II)-aggregates similar to those of Cd4-alphaMT, where the His residue plays the role of the substituted Cys. Despite the structural implications of the Cys-to-His replacement, the dissociation constants showed no major decrease in the Cd-binding affinity in any of the mutants assayed compared with the wild-type.

Classical molecular interaction potentials: improved setup procedure in molecular dynamics simulations of proteins.

The latest version of the classical molecular interaction potential (CMIP) has the ability to predict the position of crystallographic waters in several proteins with great accuracy. This article analyzes the ability of the CMIP functional to improve the setup procedure of the molecular system in molecular dynamics (MD) simulations of proteins. To this end, the CMIP strategy is used to include both water molecules and counterions in different protein systems. The structural details of the configurations sampled from trajectories obtained using the CMIP setup procedure are compared with those obtained from trajectories derived from a standard equilibration process. The results show that standard MD simulations can lead to artifactual results, which are avoided using the CMIP setup procedure. Because the CMIP is easy to implement at a low computational cost, it can be very useful in obtaining reliable MD trajectories.

Sequential amino acid exchange across b(0,+)-like system in chicken brush border jejunum.

In the small intestine, cationic amino acids are transported by y(+)-like and b(0,+)-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b(0,+)-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b(0,+)-like system functions for L-arginine as an obligatory exchanger since its transport capacity increases 100-200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Argin efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Argout is higher than that of Argin, suggesting a possible order of binding (Argout first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed.

The carboxy-terminal region of human lipoprotein lipase is necessary for its exit from the endoplasmic reticulum.

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along the secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.

D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus. I. Kinetic mechanism and pH dependence of kinetic parameters, coenzyme binding and substrate inhibition.

The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD+ plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD+-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH-->NAD+ direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast. The release of NAD+ at pH 8.0 is 200-times faster than Kcat (NADH-->NAD+) whereas the release of NADH is only 5-times faster than Kcat (NAD+-->NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E-NADH complex) and < or = 6.4 (in the E-NAD+ complex) which may be the proton donor and acceptor for the chemical reaction. At pH 6.5 the mechanism changes in the NADH-->NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+. In addition the inhibition by excess oxo-4-methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids.

Obligatory amino acid exchange via systems bo,+-like and y+L-like. A tertiary active transport mechanism for renal reabsorption of cystine and dibasic amino acids.

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the "renal proximal tubular" cell line OK. System bo, +-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.