Gene preference in maple syrup urine disease.

Untreated maple syrup urine disease (MSUD) results in mental and physical disabilities and often leads to neonatal death. Newborn-screening programs, coupled with the use of protein-modified diets, have minimized the severity of this phenotype and allowed affected individuals to develop into productive adults. Although inheritance of MSUD adheres to rules for single-gene traits, mutations in the genes for E1alpha, E1beta, or E2 of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex can cause the disease. Randomly selected cell lines from 63 individuals with clinically diagnosed MSUD were tested by retroviral complementation of branched-chain alpha-ketoacid dehydrogenase activity to identify the gene locus for mutant alleles. The frequencies of the mutations were 33% for the E1alpha gene, 38% for the E1beta gene, and 19% for the E2 gene. Ten percent of the tested cell lines gave ambiguous results by showing no complementation or restoration of activity with two gene products. These results provide a means to establish a genotype/phenotype relationship in MSUD, with the ultimate goal of unraveling the complexity of this single-gene trait. This represents the largest study to date providing information on the genotype for MSUD.

Controlled overexpression of BCKD kinase expression: metabolic engineering applied to BCAA metabolism in a mammalian system.

A common metabolic complication of human disease is uncontrolled muscle protein breakdown or cachexia, which occurs in patients with chronic diseases such as cancer, AIDS, renal failure, and diabetes. Increased branched-chain amino acid catabolism is implicated as causal and has stimulated the investigation of methods to regulate the metabolism of these amino acids. Here we demonstrate doxycycline-controlled overexpression of a branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase transgene in mammalian cell culture. This kinase functions to inactivate the BCKD complex by phosphorylation, thus preventing the catabolism of these essential, regulatory metabolites. In this study, doxycycline treatment leads to a 10-fold increase in BCKD kinase protein. The transgene-generated kinase is rapidly incorporated within mitochondria and functions correctly to inactivate the BCKD complex. The maximum reduction in basal BCKD activity achieved was 94%. Unexpectedly, total BCKD activity was also decreased by kinase overexpression despite no observable change in expression of the BCKD catalytic proteins. These results demonstrate that artificial regulation of branched-chain amino acid metabolism is possible through the controlled overexpression of a single endogenous enzyme and suggest the feasibility of clinical applications.

Amino acid deprivation induces translation of branched-chain alpha-ketoacid dehydrogenase kinase.

Leucine, isoleucine, and valine are used by cells for protein synthesis or are catabolized into sources for glucose and lipid production. These branched-chain amino acids influence proteolysis, hormone release, and cell cycle progression along with their other metabolic roles. The branched-chain amino acids play a central role in regulating cellular protein turnover by reducing autophagy. These essential amino acids are committed to their catabolic fate by the activity of the branched-chain alpha-ketoacid dehydrogenase complex. Activity of the branched-chain alpha-ketoacid dehydrogenase complex is regulated by phosphorylation/inactivation of the alpha-subunit performed by a complex specific kinase. Here we show that elimination of the branched-chain amino acids from the medium of cultured cells results in a two- to threefold increased production of the branched-chain alpha-ketoacid dehydrogenase kinase with a decrease in the activity state of the branched-chain alpha-ketoacid dehydrogenase complex. The mechanism cells use to increase kinase production under these conditions involves recruitment of the kinase mRNA into polyribosomes. Promoter activity and the steady-state concentration of the mRNA are unchanged by these conditions.

Import rate of the E1beta subunit of human branched chain alpha-ketoacid dehydrogenase is a limiting factor in the amount of complex formed in the mitochondria.

Components of the mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex are all encoded by nuclear genes. The functional complex is formed with a known stoichiometric relationship of subunits, but how they enter the mitochondria and form the complex is not defined. Although cytosolic precursors for several of the proteins have been identified, the requirements for import and processing have not been described. Here we demonstrate the similar requirements for in vitro import and processing of the three catalytic subunits unique the this complex. Import was not affected by the amount of endogenous BCKD within the mitochondria. No cooperativity or competition among the subunits for import was found when subunits were used in combination. The relative rates of entry are E1alpha>E2>/=E1beta, making E1beta the limiting component supporting previously reported observations.

Human mutations affecting branched chain alpha-ketoacid dehydrogenase.

Maple syrup urine disease results from defective function of the branched chain alpha-ketoacid dehydrogenase complex [BCKD] within the matrix of the mitochondria. This disorder in humans is inherited as an autosomal recessive trait with an incidence of 1 in 150,000 live-births in the general population and 1/176 for the Mennonite population. Over 50 different causal mutations are known to exist scattered among the three genes unique to the catalytic function of the enzyme complex. The defect was first described in 1954 and much has been learned about the genes and proteins involved in this rare human disorder. The enzyme is present in all mammalian cells that contain mitochondria, and the activity of BCKD is regulated by phosphorylation through a complex-specific kinase. Expression of the kinase is regulated by metabolic and hormonal components. Naturally occurring mutations are used to define the molecular mechanisms of transcription, translation, protein import into mitochondria and the assembly of the component proteins into a functional complex. The long-term pathophysiology of BCKD dysfunction remains to be explained. What began as a focused interest in BCKD due to the associated disease, has broadened into a quest to understand the role of BCKD in regulation of leucine levels and in turn controlling protein metabolism and hormone release.

Murine branched chain alpha-ketoacid dehydrogenase kinase; cDNA cloning, tissue distribution, and temporal expression during embryonic development.

These studies were designed to demonstrate the structural and functional similarity of murine branched chain alpha-ketoacid dehydrogenase and its regulation by the complex-specific kinase. Nucleotide sequence and deduced amino acid sequence for the kinase cDNA demonstrate a highly conserved coding sequence between mouse and human. Tissue-specific expression in adult mice parallels that reported in other mammals. Kinase expression in female liver is influenced by circadian rhythm. Of special interest is the fluctuating expression of this kinase during embryonic development against the continuing increase in the catalytic subunits of this mitochondrial complex during development. The need for regulation of the branched chain alpha-ketoacid dehydrogenase complex by kinase expression during embryogenesis is not understood. However, the similarity of murine branched chain alpha-ketoacid dehydrogenase and its kinase to the human enzyme supports the use of this animal as a model for the human system.

Two new mutations in the human E1 beta subunit of branched chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defective function of the mitochondrial branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in both alleles of any of three genes for component proteins result in the clinical phenotype. Two discrete mutant alleles for the E1 beta subunit of the decarboxylase component in a proband with MSUD are defined and parental origin of each allele identified. The maternal mutation, an A to T transversion at nucleotide 526 in the coding sequence, potentiates an asparagine to tyrosine change at position 126 (N126Y). The paternal mutant allele contains a C to T transition at nucleotide 970 introducing a stop codon (R274 ). Western blot analysis revealed a 75% reduction in the E1 beta-N126Y protein and an absence of the R274* truncated protein in proband cells. Both mutant proteins could be synthesized, imported into mitochondria, and processed in vitro. Functional analysis of the mutant proteins provided new information on the role of E1 beta in the activity of BCKD. In vivo the E1 beta-N126Y protein associated into macromolecular complexes indistinguishable from those formed with the wild type E1 beta protein. However, catalytic activity of these complexes in proband cells was < 1% of wild type activity. Alignment comparisons with other thiamin pyrophosphate-requiring enzymes suggests the N126Y substitution could interfere with interactions of the protein with the cofactor causing inactivity. The truncated E1 beta-R274* protein is unstable and not found in mitochondria from the patient derived cells.

A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae.

The metabolism of leucine to isoamyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy. The product of leucine transamination, alpha-ketoisocaproate had four potential routes to isoamyl alcohol. The first, via branched-chain alpha-keto acid dehydrogenase to isovaleryl-CoA with subsequent conversion to isovalerate by acyl-CoA hydrolase operates in wild-type cells where isovalerate appears to be an end product. This pathway is not required for the synthesis of isoamyl alcohol because abolition of branched-chain alpha-keto acid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isoamyl alcohol. A second possible route was via pyruvate decarboxylase; however, elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant did not decrease the levels of isoamyl alcohol produced. A third route utilizes alpha-ketoisocaproate reductase (a novel activity in Saccharomyces cerevisiae) but with no role in the formation of isoamyl alcohol from alpha-hydroxyisocaproate because cell homogenates could not convert alpha-hydroxyisocaproate to isoamyl alcohol. The final possibility was that a pyruvate decarboxylase-like enzyme encoded by YDL080c appears to be the major route of decarboxylation of alpha-ketoisocaproate to isoamyl alcohol although disruption of this gene reveals that at least one other unidentified decarboxylase can substitute to a minor extent.

Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase.

Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate alpha-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the alpha-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.

Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex.

Constitutive expression of nuclear genes encoding mitochondrial proteins raises the question of whether these proteins are present in similar amounts in mitochondria of different tissues. We report that amounts of a single multienzyme complex can vary on a per mitochondrion basis depending on the number of mitochondria per cell. Human branched-chain alpha-keto acid dehydrogenase (BCKD) expression is used as a paradigm in these studies. Expression is compared and contrasted in HepG2 and DG75 cells in which mitochondrial content is twofold higher in the hepatocarcinoma line than in the lymphoblastoid line. Per cell, BCKD activity is equal in the two cells types, but BCKD protein concentration per mitochondrion is twofold higher in DG75 cells. Steady-state mRNA levels do not appear to be directly related to amounts of protein in the two cell lines. To test whether one subunit is limiting in formation of complex, overexpression of each BCKD subunit was elicited by plasmid transfection of the DG75 cells. Only overexpression of the beta-subunit of the decarboxylase component induced more BCKD activity without apparent increase in mRNA for the other endogenously expressed subunits. This implies that free BCKD subunits exist in a cell and can be recruited into an active complex when the limiting subunit becomes available.

Autoantibodies to BCOADC-E2 in patients with primary biliary cirrhosis recognize a conformational epitope.

Primary biliary cirrhosis (PBC) is an autoimmune disease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoantigens recognized by autoantibodies in PBC are members of the 2-oxo-acid dehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate dehydrogenase complex (PDC-E2) and the E2 component of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2). The autoantibody response to PDC-E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational components. The presence of a single immunodominant epitope in PDC-E2 is unusual when contrasted to the immune response to autoantigens in other human autoimmune diseases. We have mapped the epitope recognized by antimitochondrial autoantibodies (AMA) specific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 complementary DNA (cDNA) and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, enzyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the absence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid.

Rat muscle branched-chain ketoacid dehydrogenase activity and mRNAs increase with extracellular acidemia.

The rate-limiting enzyme in branched-chain amino acid catabolism is branched-chain ketoacid dehydrogenase (BCKAD). In rats fed NH4Cl to induce acidemia, we find increased basal BCKAD activity as well as maximal activity in skeletal muscle. Concurrently, there is a > 10-fold increase in mRNAs of BCKAD subunits in skeletal muscle plus an increase in cardiac muscle but not in liver or kidney. There was no increase in mRNA for malate dehydrogenase or for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Evaluation of the translation capacity of BCKAD mRNAs in muscle of acidemic rats yielded more immunoreactive BCKAD whether the proteins were synthesized from muscle RNA using rabbit reticulocyte lysate or directly using postmitochondrial homogenates. Although the RNA from muscle of acidemic rats yielded twice as much BCKAD protein, we found no net increase in mitochondrial BCKAD protein in muscle by Western blotting. Because there is increased proteolysis in muscle of rats with acidemia, the increase in mRNA might be a mechanism to augment BCKAD synthesis and activity in muscle.

Epitope mapping of the branched chain alpha-ketoacid dehydrogenase dihydrolipoyl transacylase (BCKD-E2) protein that reacts with sera from patients with idiopathic dilated cardiomyopathy.

Sera from 29 of 48 patients with idiopathic dilated cardiomyopathy (IDCM) and six of six patients with dilated cardiomyopathy (DCM) secondary to suspected viral myocarditis were shown to react with the branched chain alpha-ketoacid dehydrogenase (BCKD) complex mitochondrial proteins. Whereas sera from only 1 of 26 patients with ischemic heart disease showed reactivity against the BCKD complex protein, 0 of 30 sera from normal human volunteers, 0 of 64 sera from patients with lupus, and 0 of 34 sera from patients with rheumatoid arthritis showed detectable reactivity, denoting an element of specificity for the reactivity of sera from IDCM patients. The major reactivity was localized to the dihydrolipoyl transacylase (E2) component of BCKD complex. By using recombinant techniques, the immunodominant BCKD-E2 epitope recognized by sera from IDCM patients was localized to amino acid (aa) sequences 116 to 134. Each of the IDCM sera that reacted with the native BCKD complex was shown to react with the immunodominant peptide, as defined by a peptide inhibition ELISA and by an ELISA using the reactive peptide conjugated to BSA. Sera from IDCM patients that reacted with the native BCKD complex and the reactive peptide also showed inhibition of BCKD enzyme activity. The possible mechanisms for the induction of the Abs and the implications of these findings for the pathogenesis of IDCM are discussed.

Molecular genetic characterization of maple syrup urine disease in European families.

Maple syrup urine disease results from defects in the branched chain alpha-ketoacid dehydrogenase complex. Cells from seven German, three Turkish, and two Italian families including five consanguineous matings were analyzed for the causative mutations. Enzyme assays were used to confirm the initial clinical diagnosis of all probands. Immunoblots of mitochondrial proteins from these probands revealed reduced expression of the E1 alpha and beta proteins of the complex. Previous studies showed that interaction of alpha and beta was necessary to stabilize both proteins so that defects in either protein can result in decreased presence of both. The E1 alpha Y393N mutation common in the Mennonite population that results in diminished amounts of both alpha and beta proteins was not the cause of the reduction in these European patients.

Comparative metabolism and structure of BCKD-E2 in primary biliary cirrhosis.

The identification and cloning of the mitochondrial autoantigens in primary biliary cirrhosis (PBC) have provided new clues in disease pathogenesis. The two major autoantigens are the E2 subunits of pyruvate dehydrogenase and branched-chain ketoacid dehydrogenase (BCKD). Interestingly, one of these complexes, BCKD-E2, is already well known to clinical medicine based on its association with genetic mutations in maple syrup urine disease (MSUD). Patients with this disease have an inability to metabolize branched-chain amino acids. In the present study, we have taken advantage of the known sequence of BCKD-E2 from normal humans, and addressed the issue of whether there is an altered autoantigen sequence in hepatocytes of individuals with primary biliary cirrhosis. In particular, we examined both the leader sequence and the B-cell immunodominant epitope, the lipoic acid domain. In addition, because patients with PBC have autoantibodies to the BCKD-E2 complex, we have quantitated plasma levels of alpha-ketoacids potentially affected in maple syrup urine disease. These include pyruvic acid (PY), phenylpyruvic acid (PP), alpha-ketoisocaproic acid (KIC) alpha-ketoisovalerate (KIV) and alpha-keto-beta-methylvaleric acid (KMV). The levels of these alpha-ketoacids were compared in patients with primary sclerosing cholangitis and normal volunteers. The sequence of BCKD-E2 obtained from PBC hepatocytes showed homology with normal BCKD. Further studies of autoantigen structure and sequence are clearly indicated, including those involved in mitochondrial transport and localization. Finally, we noted a statistically significant increase in all plasma alpha-ketoacids except alpha-keto-beta-methylvaleric acid in PBC patients.(ABSTRACT TRUNCATED AT 250 WORDS)