Dehydrogenase reductase 9 (SDR9C4) and related homologs recognize a broad spectrum of lipid mediator oxylipins as substrates.

Bioactive oxylipins play multiple roles during inflammation and in the immune response, with termination of their actions partly dependent on the activity of yet-to-be characterized dehydrogenases. Here, we report that human microsomal dehydrogenase reductase 9 (DHRS9, also known as SDR9C4 of the short-chain dehydrogenase/reductase (SDR) superfamily) exhibits a robust oxidative activity toward oxylipins with hydroxyl groups located at carbons C9 and C13 of octadecanoids, C12 and C15 carbons of eicosanoids, and C14 carbon of docosanoids. DHRS9/SDR9C4 is also active toward lipid inflammatory mediator dihydroxylated Leukotriene B4 and proresolving mediators such as tri-hydroxylated Resolvin D1 and Lipoxin A4, although notably, with lack of activity on the 15-hydroxyl of prostaglandins. We also found that the SDR enzymes phylogenetically related to DHRS9, i.e., human SDR9C8 (or retinol dehydrogenase 16), the rat SDR9C family member known as retinol dehydrogenase 7, and the mouse ortholog of human DHRS9 display similar activity toward oxylipin substrates. Mice deficient in DHRS9 protein are viable, fertile, and display no apparent phenotype under normal conditions. However, the oxidative activity of microsomal membranes from the skin, lung, and trachea of Dhrs9-/- mice toward 1 µM Leukotriene B4 is 1.7- to 6-fold lower than that of microsomes from wild-type littermates. In addition, the oxidative activity toward 1 µM Resolvin D1 is reduced by about 2.5-fold with DHRS9-null microsomes from the skin and trachea. These results strongly suggest that DHRS9 might play an important role in the metabolism of a wide range of bioactive oxylipins in vivo.

Characterization of subunit interactions in the hetero-oligomeric retinoid oxidoreductase complex.

The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa. The C-terminal residues 317-331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291-295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.

Changes in retinoid metabolism and signaling associated with metabolic remodeling during fasting and in type I diabetes.

Liver is the central metabolic hub that coordinates carbohydrate and lipid metabolism. The bioactive derivative of vitamin A, retinoic acid (RA), was shown to regulate major metabolic genes including phosphoenolpyruvate carboxykinase, fatty acid synthase, carnitine palmitoyltransferase 1, and glucokinase among others. Expression levels of these genes undergo profound changes during adaptation to fasting or in metabolic diseases such as type 1 diabetes (T1D). However, it is unknown whether the levels of hepatic RA change during metabolic remodeling. This study investigated the dynamics of hepatic retinoid metabolism and signaling in the fed state, in fasting, and in T1D. Our results show that fed-to-fasted transition is associated with significant decrease in hepatic retinol dehydrogenase (RDH) activity, the rate-limiting step in RA biosynthesis, and downregulation of RA signaling. The decrease in RDH activity correlates with the decreased abundance and altered subcellular distribution of RDH10 while Rdh10 transcript levels remain unchanged. In contrast to fasting, untreated T1D is associated with upregulation of RA signaling and an increase in hepatic RDH activity, which correlates with the increased abundance of RDH10 in microsomal membranes. The dynamic changes in RDH10 protein levels in the absence of changes in its transcript levels imply the existence of posttranscriptional regulation of RDH10 protein. Together, these data suggest that the downregulation of hepatic RA biosynthesis, in part via the decrease in RDH10, is an integral component of adaptation to fasting. In contrast, the upregulation of hepatic RA biosynthesis and signaling in T1D might contribute to metabolic inflexibility associated with this disease.

Generation of Retinaldehyde for Retinoic Acid Biosynthesis.

The concentration of all-trans-retinoic acid, the bioactive derivative of vitamin A, is critically important for the optimal performance of numerous physiological processes. Either too little or too much of retinoic acid in developing or adult tissues is equally harmful. All-trans-retinoic acid is produced by the irreversible oxidation of all-trans-retinaldehyde. Thus, the concentration of retinaldehyde as the immediate precursor of retinoic acid has to be tightly controlled. However, the enzymes that produce all-trans-retinaldehyde for retinoic acid biosynthesis and the mechanisms responsible for the control of retinaldehyde levels have not yet been fully defined. The goal of this review is to summarize the current state of knowledge regarding the identities of physiologically relevant retinol dehydrogenases, their enzymatic properties, and tissue distribution, and to discuss potential mechanisms for the regulation of the flux from retinol to retinaldehyde.

Mice lacking the epidermal retinol dehydrogenases SDR16C5 and SDR16C6 display accelerated hair growth and enlarged meibomian glands.

Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.

Tissue-specific kinase expression and activity regulate flux through the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex (PDC) is a multienzyme assembly that converts pyruvate to acetyl-CoA. As pyruvate and acetyl-CoA play central roles in cellular metabolism, understanding PDC regulation is pivotal to understanding the larger metabolic network. The activity of mammalian PDC is regulated through reversible phosphorylation governed by at least four isozymes of pyruvate dehydrogenase kinase (PDK). Deciphering which kinase regulates PDC in organisms at specific times or places has been challenging. In this study, we analyzed mouse strains carrying targeted mutations of individual isozymes to explore their role in regulating PDC activity. Analysis of protein content of PDK isozymes in major metabolic tissues revealed that PDK1 and PDK2 were ubiquitously expressed, whereas PDK3 and PDK4 displayed a rather limited tissue distribution. Measurement of kinase activity showed that PDK1 is the principal isozyme regulating hepatic PDC. PDK2 was largely responsible for inactivation of PDC in tissues of muscle origin and brown adipose tissue (BAT). PDK3 was the principal kinase regulating pyruvate dehydrogenase activity in kidney and brain. In a well-fed state, the tissue levels of PDK4 protein were fairly low. In most tissues tested, PDK4 ablation had little effect on the overall rates of inactivation of PDC in kinase reaction. Taken together, these data strongly suggest that the activity of PDC is regulated by different isozymes in different tissues. Furthermore, it appears that the overall flux through PDC in a given tissue largely reflects the properties of the PDK isozyme that is principally responsible for the regulation of PDC activity in that tissue.

Allosteric coupling in pyruvate dehydrogenase kinase 2.

Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.

Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.

PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect the L2- and E1-binding sites. Significant differences are found between PDHK2 and PDHK3 with respect to the structure of their lipoyllysine-binding cavities, providing the first structural support to a number of studies showing that these isozymes are markedly different with respect to their affinity for the L2 domain. Both structures display a novel type II potassium-binding site located on the PDHK2 interface with the L2 domain. Binding of potassium ion at this site rigidifies the interface and appears to be critical in determining the strength of L2 binding. Evidence is also presented that potassium ions are indispensable for the cross-talk between the nucleotide- and L2-binding sites of PDHK2. The latter is believed to be essential for the movement of PDHK2 along the surface of the transacetylase scaffold.

Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2.

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.

Amino acid residues responsible for the recognition of dichloroacetate by pyruvate dehydrogenase kinase 2.

Dichloroacetate (DCA) is a promising anticancer and antidiabetic compound targeting the mitochondrial pyruvate dehydrogenase kinase (PDHK). This study was undertaken in order to map the DCA-binding site of PDHK2. Here, we present evidence that R114, S83, I157 and, to some extent, H115 are essential for DCA binding. We also show that Y80 and D117 are required for the communication between the DCA-binding site and active site of PDHK2. These observations provide important insights into the mechanism of DCA action that may be useful for the design of new, more potent therapeutic compounds.

Identification of a novel PP2C-type mitochondrial phosphatase.

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.

Gender and post-ischemic recovery of hypertrophied rat hearts.

BACKGROUND: Gender influences the cardiac response to prolonged increases in workload, with differences at structural, functional, and molecular levels. However, it is unknown if post-ischemic function or metabolism of female hypertrophied hearts differ from male hypertrophied hearts. Thus, we tested the hypothesis that gender influences post-ischemic function of pressure-overload hypertrophied hearts and determined if the effect of gender on post-ischemic outcome could be explained by differences in metabolism, especially the catabolic fate of glucose. METHODS: Function and metabolism of isolated working hearts from sham-operated and aortic-constricted male and female Sprague-Dawley rats before and after 20 min of no-flow ischemia (N = 17 to 27 per group) were compared. Parallel series of hearts were perfused with Krebs-Henseleit solution containing 5.5 mM [5-3H/U-14C]-glucose, 1.2 mM [1-14C]-palmitate, 0.5 mM [U-14C]-lactate, and 100 mU/L insulin to measure glycolysis and glucose oxidation in one series and oxidation of palmitate and lactate in the second. Statistical analysis was performed using two-way analysis of variance. The sequential rejective Bonferroni procedure was used to correct for multiple comparisons and tests. RESULTS: Female gender negatively influenced post-ischemic function of non-hypertrophied hearts, but did not significantly influence function of hypertrophied hearts after ischemia such that mass-corrected hypertrophied heart function did not differ between genders. Before ischemia, glycolysis was accelerated in hypertrophied hearts, but to a greater extent in males, and did not differ between male and female non-hypertrophied hearts. Glycolysis fell in all groups after ischemia, except in non-hypertrophied female hearts, with the reduction in glycolysis after ischemia being greatest in males. Post-ischemic glycolytic rates were, therefore, similarly accelerated in hypertrophied male and female hearts and higher in female than male non-hypertrophied hearts. Glucose oxidation was lower in female than male hearts and was unaffected by hypertrophy or ischemia. Consequently, non-oxidative catabolism of glucose after ischemia was lowest in male non-hypertrophied hearts and comparably elevated in hypertrophied hearts of both sexes. These differences in non-oxidative glucose catabolism were inversely related to post-ischemic functional recovery. CONCLUSION: Gender does not significantly influence post-ischemic function of hypertrophied hearts, even though female sex is detrimental to post-ischemic function in non-hypertrophied hearts. Differences in glucose catabolism may contribute to hypertrophy-induced and gender-related differences in post-ischemic function.

The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes.

Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity.

The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.

Probing a putative active site of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) by site-directed mutagenesis.

The catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) is a magnesium-dependent protein phosphatase that regulates the activity of mammalian pyruvate dehydrogenase complex. Based on the sequence analysis, it was hypothesized that PDP1c is related to the mammalian magnesium-dependent protein phosphatase type 1, with Asp54, Asp347, and Asp445 contributing to the binuclear metal-binding center, and Asn49 contributing to the phosphate-binding sites. In this study, we analyzed the functional significance of these amino acid residues using a site-directed mutagenesis. It was found that substitution of each of these residues had a significant impact on PDP1c activity toward the protein substrate. The activities of Asp54, Asp347, and Asp445 mutants were decreased more than 1000-fold. The activity of Asn49 mutant was 2.5-fold lower than the activity of wild-type PDP1c. The decrease in activity of Asp54 and Asp347 came about, most likely, as a result of impaired magnesium binding. Unexpectedly, it was found that the Asp445 mutant bound Mg(2+) ions similarly to the wild-type enzyme. Accordingly, the Asp445 mutant was found to be active with the artificial substrate p-nitrophenyl phosphate (pNPP). Asp54 and Asp347 mutants did not demonstrate any appreciable activity with pNPP. Together, these observations strongly suggest that Asn49, Asp54, and Asp347 are important for the catalysis of the phosphatase reaction, contributing to the phosphate- and metal-binding centers of PDP1c. In contrast, Asp445 is not required for catalysis. The exact role of Asp445 remains to be established, but indirect evidence suggests that it might be involved in the control of interactions between PDP1c and the protein substrate pyruvate dehydrogenase.

Regular exercise is associated with a protective metabolic phenotype in the rat heart.

Adaptation of myocardial energy substrate utilization may contribute to the cardioprotective effects of regular exercise, a possibility supported by evidence showing that pharmacological metabolic modulation is beneficial to ischemic hearts during reperfusion. Thus we tested the hypothesis that the beneficial effect of regular physical exercise on recovery from ischemia-reperfusion is associated with a protective metabolic phenotype. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured in isolated working hearts from sedentary control (C) and treadmill-trained (T: 10 wk, 4 days/wk) female Sprague-Dawley rats submitted to 20 min ischemia and 40 min reperfusion. Training resulted in myocardial hypertrophy (1.65 +/- 0.05 vs. 1.30 +/- 0.03 g heart wet wt, P < 0.001) and improved recovery of function after ischemia by nearly 50% (P < 0.05). Glycolysis was 25-30% lower in T hearts before and after ischemia (P < 0.05), whereas rates of glucose oxidation were 45% higher before ischemia (P < 0.01). As a result, the fraction of glucose oxidized before and after ischemia was, respectively, twofold and 25% greater in T hearts (P < 0.05). Palmitate oxidation was 50-65% greater in T than in C before and after ischemia (P < 0.05), whereas lactate oxidation did not differ between groups. Alteration in content of selected enzymes and proteins, as assessed by immunoblot analysis, could not account for the reduction in glycolysis or increase in glucose and palmitate oxidation observed. Combined with the studies on the beneficial effect of pharmacological modulation of energy metabolism, the present results provide support for a role of metabolic adaptations in protecting the trained heart against ischemia-reperfusion injury.

Characterization of the isozymes of pyruvate dehydrogenase phosphatase: implications for the regulation of pyruvate dehydrogenase activity.

The activity of mammalian pyruvate dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of pyruvate dehydrogenase phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the alpha chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of pyruvate dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of pyruvate dehydrogenase.

Starvation and diabetes reduce the amount of pyruvate dehydrogenase phosphatase in rat heart and kidney.

The pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes to conserve three-carbon compounds for gluconeogenesis. This is achieved by an increase in the extent of PDC phosphorylation caused in part by increased pyruvate dehydrogenase kinase (PDK) activity due to increased PDK expression. This study examined whether altered pyruvate dehydrogenase phosphatase (PDP) expression also contributes to changes in the phosphorylation state of PDC during starvation and diabetes. Of the two PDP isoforms expressed in mammalian tissues, the Ca(2+)-sensitive isoform (PDP1) is highly expressed in rat heart, brain, and testis and is detectable but less abundant in rat muscle, lung, kidney, liver, and spleen. The Ca(2+)-insensitive isoform (PDP2) is abundant in rat kidney, liver, heart, and brain and is detectable in spleen and lung. Starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. These findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes.

Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general. In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4). These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated. Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell. The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography. The purified kinase was stable and was not subjected to reassortment of the subunits. The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation. These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity.

Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex.

Protein-protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1-PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3-L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10 microM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1=PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.