Novel functions of the alpha-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H(2)O(2) and to probe the mechanism underlying these changes. Since the response to H(2)O(2) is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H(2)O(2) decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H(2)O(2) inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H(2)O(2) converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H(2)O(2) diminished glutathione to a similar level after 24 h. However, H(2)O(2), but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H(2)O(2) together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.     

Mild reduction in the activity of the α-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis

Diminished energy metabolism and reduced activity of brain α-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-¹³C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-¹³C]glucose, the E2k67 cells showed a greater increase in ¹³C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased ¹³C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.     

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.     

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.     

Immunochemical Characterization of the Deficiency of the α-Ketoglutarate Dehydrogenase Complex in Thiamine-Deficient Rat Brain

Abstract: Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.     

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.     

Phosphonate analogues of alpha-ketoglutarate inhibit the activity of the alpha-ketoglutarate dehydrogenase complex isolated from brain and in cultured cells

The alpha-ketoglutarate dehydrogenase complex (KGDHC), a control point of the tricarboxylic acid cycle, is partially inactivated in brain in many neurodegenerative diseases. Potent and specific KGDHC inhibitors are needed to probe how the reduced KGDHC activity alters brain function. Previous studies showed that succinyl phosphonate (SP) effectively inhibits muscle and Escherichia coli KGDHC [Biryukov, A. I., Bunik, V. I., Zhukov, Yu. N., Khurs, E. N., and Khomutov, R. M. (1996) FEBS Lett. 382, 167-170]. To identify the phosphonates with the highest affinity toward brain KGDHC and with the greatest effect in living cells, we investigated the ability of SP and several of its ethyl esters to inhibit brain KGDHC, other alpha-keto acid-dependent enzymes, and KGDHC in intact cells. At a concentration of 0.01 mM, SP and its phosphonoethyl (PESP) and carboxyethyl (CESP) esters completely inhibited isolated brain KGDHC even in the presence of a 200-fold higher concentration of its substrate [alpha-ketoglutarate (KG)], while the diethyl (DESP) and triethyl (TESP) esters were ineffective. In cultured human fibroblasts, 0.01 mM SP, PESP, or CESP produced 70% inhibition of KGDHC. DESP and TESP were also inhibitory in the cell system, but only after preincubation, suggesting the release of their charged groups by cellular esterases. Thus, SP and its monoethyl esters target cellular KGDHC directly, while the di- and triethyl esters are activated in intact cells. When tested on other enzymes that bind KG or related alpha-keto acids, SP had minimal effects and its two esters (CESP and TESP) were ineffective even at a concentration (0.1 mM) 1 order of magnitude higher than that which inhibited cellular KGDHC activity. The high specificity in targeting KGDHC, penetration into cells, and minimal transformation by cellular enzymes indicate that SP and its esters should be useful in studying the effects of reduced KGDHC activity on neuronal and brain function.     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex

Nuclear genes coding for the M_r 17000, 14000 and 11000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay. This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell. A first characterization of the clones is presented. The genes are not closely linked in the genome and those coding for M_r 14000 and 11000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.     

Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2. It is known that the M2 subunit plays a role in tumorgenicity and metastasis. In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418. The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG). However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by 14CDP reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG. M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours. Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.     

Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells

We previously reported stable transfection of estrogen receptor alpha (ERalpha) into the ER-negative MDA-MB-231 cells (S30) as a tool to examine the mechanism of action of estrogen and antiestrogens [J. Natl. Cancer Inst. 84 (1992) 580]. To examine the mechanism of ERbeta action directly, we have similarly created ERbeta stable transfectants in MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with ERbeta cDNA and clones were screened by estrogen response element (ERE)-luciferase assay and ERbeta mRNA expression was quantified by real-time RT-PCR. Three stable MDA-MB-231/ERbeta clones were compared with S30 cells with respect to their growth properties, ability to activate ERE- and activating protein-1 (AP-1) luciferase reporter constructs, and the ability to activate the endogenous ER-regulated transforming growth factor alpha (TGFalpha) gene. ERbeta6 and ERbeta27 clones express 300-400-fold and the ERbeta41 clone express 1600-fold higher ERbeta mRNA levels compared with untransfected MDA-MB-231 cells. Unlike S30 cells, 17beta-estradiol (E2) does not inhibit ERbeta41 cell growth. ERE-luciferase activity is induced six-fold by E2 whereas neither 4-hydroxytamoxifen (4-OHT) nor ICI 182, 780 activated an AP-1-luciferase reporter. TGFalpha mRNA is induced in response to E2, but not in response to 4-OHT. MDA-MB-231/ERbeta clones exhibit distinct characteristics from S30 cells including growth properties and the ability to induce TGFalpha gene expression. Furthermore, ERbeta, at least in the context of the MDA-MB-231 cellular milieu, does not enhance AP-1 activity in the presence of antiestrogens. In summary, the availability of both ERalpha and ERbeta stable breast cancer cell lines now allows us to compare and contrast the long-term consequences of individual signal transduction pathways.     

Sequence and expression of human protein kinase C-epsilon.

Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.     

The alpha-ketoglutarate dehydrogenase complex in neurodegeneration

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.     

The α-ketoglutarate-dehydrogenase complex

Damage from oxidative stress and mitochondrial dysfunction occur together in many common neurodegenerative diseases. The enzymes that form the mitochondrial alpha-ketoglutarate- dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme system of the tricarboxylic acid cycle, might mediate the interaction of these processes. KGDHC activity is reduced in numerous age-related neurodegenerative diseases and is diminished by oxidative stress. In Alzheimer's disease (AD), the reduction correlates highly to diminished mental performance. Thus, research has focused on the mechanisms by which select oxidants reduce KGDHC and the consequences of such a reduction. Diminished KGDHC in cells is associated with apoptosis without changes in the mitochondrial membrane potential. Studies of isolated mitochondria and of animal models suggest that a reduction in KGDHC can predispose to damage by other toxins that promote neurodegeneration. Diminished oxidative metabolism can be plausibly linked to pathological features of neurodegenerative diseases (e.g., reduced mental function, the plaques and tangles in AD). Thus, reductions in KGDHC might be central to the pathophysiology of these diseases. Studies of proteins, cells, animal models, and humans suggest that treatments to diminish, or bypass, the reduction in KGDHC might be beneficial in age-related neurodegenerative disorders.     

Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells

The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91(phox), p22(phox), p47(phox), and p67(phox), in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91(phox) and p22(phox). After subcellular protein fractionation, approximately 50% of the gp91(phox) (91-kDa band), p22(phox), p67(phox), and p40(phox) pools and approximately 30% of the p47(phox) were present in the 1475 x g ("nucleus-rich") fraction. Likewise, approximately 50% of total NADPH-dependent O(2)() production (assessed by lucigenin (5 microm) chemiluminescence) was found in the 1475 x g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22(phox), gp91(phox), p47(phox), p67(phox), and p40(phox) existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton.     

Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Discoordinate synthesis of the 11-kd subunit in response to increased gene copy number

In wild-type yeast cells, steady-state concentrations of subunits of the ubiquinol-cytochrome c reductase complex (complex III) and the levels of their translatable mRNAs change coordinately in response to the need for mitochondrial function. Despite this, re-introduction of the cloned gene for one of the subunits (11 kd) into cells by transformation with a free-replicating plasmid results in the discoordinate synthesis of this subunit only, without effects on either the synthesis or degradation of the other subunits. The overproduced subunit is associated with the mitochondrial fraction, yet does not interfere with mitochondrial function, as judged by the growth of transformed cells on nonfermentable media. Quantitative analysis of both mRNA and protein levels suggests that both translational controls and elevated turnover of excess protein contribute to a partial compensation for the effects of increased gene dosage in transformed cells. These contain approximately 30 copies of the cloned gene and 15-30 times the normal level of its mRNA. Nevertheless, synthesis of the 11-kd protein is only 6- to 8-fold higher than normal, and steady-state levels are increased only 5- to 10-fold. These findings imply that synthesis of the various subunits of complex III is not tightly coupled and that for the 11-kd subunit at least, the level of mRNA is likely to be the most important means of regulating protein level. Fine-tuning may be additionally achieved by control of translation and degradation of excess protein which is not assembled in the complex.     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.