Renal injury: similarities and differences in male and female rats with the metabolic syndrome.

The metabolic syndrome is complicated by nephropathy in humans and rats, and males are more affected than females. We hypothesized that female rats had reduced expression of glomerular oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1), attendant glomerular oxidant injury, and renal inflammation. Three groups, obese males (OM), obese females (OF), and lean males (LM) of first-generation (F(1)) hybrid rats derived from the Zucker fatty diabetic (ZDF) strain and the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) were studied from 6 to 41 weeks of age. OM had severe renal oxidant injury and renal failure. Their glomeruli expressed the LOX-1, and exhibited heavier accumulation of the lipid peroxide 4-hydroxynonenal (4-HNE). OM had compromised mitochondrial enzyme function, more renal fibrosis, and vascular leakage. Younger LM, OM, and OF ZS (ZDF/SHHF F(1) hybrid rat) rats, studied from 6 to 16 weeks of age, showed that unutilized renal lipids were comparable in OM and OF, although young OM had worse nephropathy and inflammation. In conclusion, glomerular LOX-1 expression is coupled to deposits of 4-HNE and glomerulosclerosis in OM. We presume that LOX-1 enhances glomerular uptake of oxidized lipids and renal inflammation, causing greater oxidant stress and severe glomerulosclerosis. In OF, renal protection from lipid oxidants appears to be conferred by blunted glomerular LOX-1 expression and renal inflammation.

chy1, an Arabidopsis mutant with impaired beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase.

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal beta-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes beta-hydroxyisobutyryl-CoA. We demonstrated that a human beta-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes beta-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered beta-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.

New developments in our understanding of the beta-hydroxyacid dehydrogenases.

The beta-hydroxyacid dehydrogenases are a structurally conserved family of enzymes that catalyze the NAD(+) or NADP(+)-dependent oxidation of specific beta-hydroxyacid substrates like beta-hydroxyisobutyrate. These enzymes share distinct domains of amino acid sequence homology, most of which now have assigned putative functions. 6-phosphogluconate dehydrogenase and beta-hydroxyisobutyrate dehydrogenase, the most well-characterized members, both appear to be readily inactivated by chemical modifiers of lysine residues, such as 2,4,6-trinitrobenzene sulfonate (TNBS). Peptide mapping by ESI-LCMS showed that inactivation of beta-hydroxyisobutyrate dehydrogenase with TNBS occurs with the labeling of a single lysine residue, K248. This lysine residue is completely conserved in all family members and may have structural importance relating to cofactor binding. The structural framework of the beta-hydroxyacid dehydrogenase family is shared by many bacterial homologues. One such homologue from E. coli has been cloned and expressed as recombinant protein. This protein was found to have enzymatic activity characteristic of tartronate semialdehyde reductase, an enzyme required for bacterial biosynthesis of D-glycerate. A homologue from H. influenzae was also cloned and expressed as recombinant protein. This protein was active in the oxidation of D-glycerate, but showed approximately ten-fold higher activity with four carbon substrates like beta-D-hydroxybutyrate and D-threonine. This enzyme might function in H. influenzae, and other species, in the utilization of polyhydroxybutyrates, an energy storage form specific to bacteria. Cloning and characterization of these bacterial beta-hydroxyacid dehydrogenases extends our knowledge of this enzyme family.

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition.

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.

Novel beta -hydroxyacid dehydrogenases in Escherichia coli and Haemophilus influenzae.

Our laboratory has previously reported a structurally and mechanistically related family of beta-hydroxyacid dehydrogenases with significant homology to beta-hydroxyisobutyrate dehydrogenase. A large number of the members of this family are hypothetical proteins of bacterial origin with unknown identity in terms of their substrate specificities and metabolic roles. The Escherichia coli beta-hydroxyacid dehydrogenase homologue corresponding to the locus was cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein very specifically catalyzed the NAD(+)-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde, identifying this protein as a tartronate semialdehyde reductase. Further evidence for identification as tartronate semialdehyde reductase is the observation that the coding region for this protein is directly preceded by genes coding for hydroxypyruvate isomerase and glyoxylate carboligase, two enzymes that synthesize tartronate semialdehyde, producing an operon clearly designed for d-glycerate biosynthesis from tartronate semialdehyde. The single beta-hydroxyacid dehydrogenase homologue from Haemophilus influenzae was also cloned, expressed, and purified with a 6-histidine tag. This protein also catalyzed the NAD(+)-dependent oxidation of d-glycerate but was significantly more efficient in the oxidation of four-carbon beta-hydroxyacids like d-hydroxybutyrate and d-threonine. This enzyme differs from all the presently known beta-hydroxybutyrate dehydrogenases which are well established members of the short chain dehydrogenase/reductase superfamily.

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.

A molecular model of human branched-chain amino acid metabolism.

To establish an accurate molecular model of human branched-chain amino acid (BCAA) metabolism, the distribution, activity, and expression of the first 2 enzymes in the catabolic pathway--branched-chain-amino-acid aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) complex--were determined in human tissues. The same enzyme activities were measured in rat and African green monkey tissues. Overall, the activities of BCAT and BCKD were higher in rat than in human and monkey tissues; nevertheless, the ratio of the 2 activities was similar in most tissues in the 3 species. Total oxidative capacity was concentrated in skeletal muscle and liver (> 70%) with muscle having a higher proportion of the total in humans and monkeys. In humans, brain (10-20%) and kidney (8-13%) may contribute significantly to whole-body BCAA metabolism. Furthermore, in primates the high ratio of transaminase to oxidative capacity in the entire gastrointestinal tract serves to prevent loss of essential BCAA carbon and raises the possibility that the gastrointestinal tract contributes to the plasma branched-chain alpha-keto acid pool. Quantitative polymerase chain reaction was used to examine expression of human branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), the key enzyme that regulates the activity state of the human BCKD complex and human BCAT isoenzymes. To design the primers for the polymerase chain reaction, human BCKDK was cloned. BCKDK message was found in all human tissues tested, with the highest amount in human muscle. As in rats, there was ubiquitous expression of mitochondrial BCAT, whereas mRNA for the cytosolic enzyme was at or below the limit of detection outside the brain. Finally, the role of BCAA in body nitrogen metabolism is discussed.

Mitochondrial alpha-ketoacid dehydrogenase kinases: a new family of protein kinases.

Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on the E1alpha subunit of the BCKDH complex. BCKDK inhibits the dehydrogenase activity of the BCKDH complex by introducing a negative charge into the active-site pocket of the E1 component. Protein starvation of rats induces an increase in the amount of BCKDK bound to the BCKDH complex. This causes inactivation of the BCKDH complex and conserves branched-chain amino acids for protein synthesis in the protein-starved state. Expression of the different PDK isoenzymes is tissue specific, and the different PDK isoenzymes are unique with respect to kinetic parameters for ATP and ADP and sensitivity to allosteric effectors (NADH, NAD+, coenzyme A, acetyl-CoA, pyruvate, and dichloroacetate). Preliminary experiments indicate that an increased amount of PDK2 protein partly explains the increase in PDK activity that occurs in rat liver in response to chemically induced diabetes.

Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases.

Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the liver's capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.

Primary structure and tissue-specific expression of human beta-hydroxyisobutyryl-coenzyme A hydrolase.

beta-Hydroxyisobutyryl-CoA (HIBYL-CoA) hydrolase is responsible for the specific hydrolysis of HIBYL-CoA, a saline catabolite, as well as the hydrolysis of beta-hydroxypropionyl-CoA, an intermediate in a minor pathway of propionate metabolism. We have obtained the amino acid sequences of several tryptic peptides derived from purified rat liver HIBYL-CoA hydrolase, and the NH2-terminal peptize sequence was matched to the translated sequence of a human expressed sequence tag present in the data base of the IMAGE Consortium (Lawrence Livermore National Laboratory, Livermore, CA). The complete nucleotide sequence and the deduced amino acid sequence showed no similarity to the sequences of well known thioesterases but showed significant homology to the enoyl-CoA hydratase/isomerase enzyme family. The cDNA fragment corresponding to the mature (processed) protein was expressed in Escherichia coli. The purified recombinant enzyme displayed substrate specificity very similar to that of the rat enzyme and was specifically bound by polyclonal antibodies raised against purified rat liver HIBYL-CoA hydrolase. Northern and Western blot analyses with various human tissues indicated predominant expression in liver, heart, and kidney, with discrepancies occurring in the amounts of HIBYL-CoA hydrolase mRNA compared to stably expressed protein in several tissues.

Structural and mechanistic similarities of 6-phosphogluconate and 3-hydroxyisobutyrate dehydrogenases reveal a new enzyme family, the 3-hydroxyacid dehydrogenases.

Rat 3-hydroxyisobutyrate dehydrogenase exhibits significant amino acid sequence homology with 6-phosphogluconate dehydrogenase, D-phenylserine dehydrogenase from Pseudomonas syringae, and a number of hypothetical proteins encoded by genes of microbial origin. Key residues previously proposed to have roles in substrate binding and catalysis in sheep 6-phosphogluconate dehydrogenase are highly conserved in this entire family of enzymes. Site-directed mutagenesis, chemical modification, and substrate specificity studies were used to compare possible mechanistic similarities of 3-hydroxyisobutyrate dehydrogenase with 6-phosphogluconate dehydrogenase. The data suggest that 3-hydroxyisobutyrate and 6-phosphogluconate dehydrogenases may comprise, in part, a previously unrecognized family of 3-hydroxyacid dehydrogenases.

Catabolism of isobutyrate by colonocytes.

Isolated colonocytes have more capacity for the oxidation of isobutyrate and alpha-ketoisovalerate than isolated enterocytes. Both enterocytes and colonocytes express high levels of 3-hydroxyisobutyryl-CoA hydrolase, an enzyme activity important in maintaining low intracellular concentrations of methacrylyl-CoA, a common, potentially toxic intermediate in the catabolic pathways of these compounds. In spite of comparable 3-hydroxyisobutyryl-CoA hydrolase activities in both cell types, and much greater amounts of 3-hydroxyisobutyrate dehydrogenase in colonocytes than in enterocytes, only the colonocytes produced 3-hydroxyisobutyrate as an endproduct of alpha-ketoisovalerate and isobutyrate catabolism. Butyrate very effectively inhibits isobutyrate catabolism by colonocytes, most likely by competitively inhibiting activation of isobutyrate to its CoA ester. Oleate also inhibits isobutyrate catabolism, but at a site more distal than butyrate. Starvation of rats for 72 h decreased the capacity of colonocytes for butyrate but not isobutyrate catabolism. We conclude that isobutyrate could function as a carbon source for energy and anapleurosis in colonocytes under conditions of defective butyrate oxidation or low butyrate availability.

Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain alpha-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase.

Branched-chain alpha-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1 alpha subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain alpha-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased Km for alpha-ketoisovalerate, a less increased (2.8-fold) Km for alpha-ketoisocaproate, but no change in Vmax or the Km for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among alpha-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain alpha-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate.

Chemical modification and site-directed mutagenesis studies of rat 3-hydroxyisobutyrate dehydrogenase.

Rat 3-hydroxyisobutyrate dehydrogenase shares sequence homology with the short-chain alcohol dehydrogenases. Site-directed mutagenesis and chemical modifications were used to examine the roles of cysteine residues and other residues conserved in this family of enzymes. It was found that a highly conserved tyrosine residue, Y162 in 3-hydroxyisobutyrate dehydrogenase, does not function catalytically as it may in other short-chain alcohol dehydrogenases. Of the six cysteine residues present in 3-hydroxyisobutyrate dehydrogenase, only cysteine 215 was found to be critical to catalysis. C215A and C215D mutant enzymes were catalytically inactive but produced CD spectra identical to wild-type enzyme. C215S mutant enzyme displayed a lowered Vmax than wild-type enzyme, but Km values were similar to those of wild-type enzyme. The C215S mutant enzyme was inactivated by treatment with phenylmethanesulfonyl fluoride but was not inactivated by treatment with iodoacetate, whereas the wild-type enzyme was inactivated by treatment with iodoacetate but not inactivated by treatment with phenylmethanesulfonyl fluoride. The present data suggest that 3-hydroxyisobutyrate dehydrogenase differs in mechanism from other short-chain alcohol dehydrogenases studied to date and that cysteine 215 has a critical function in catalysis, possibly as a general base catalyst.