Function Of Pyruvate Dehydrogenase Complex Research Articles

Using Student Movement to Improve Understanding of the Pyruvate Dehydrogenase Complex

Students in introductory biochemistry courses often struggle to visualize the structure of large enzyme complexes and understand the mechanisms of the reactions they catalyze. One such complex, the Pyruvate Dehydrogenase Complex (PDC), catalyzes an essential step in glucose catabolism and is comprised of many copies of three enzymes that catalyze five reactions requiring five unique cofactors. To assist students in learning of the PDC reaction and mechanism, students enrolled in in‐person biochemistry participate in a group activity to act out the mechanism of the complex using instructor‐directed student movement. The effect of acting or observing the activity on learning of PDC structure, mechanism, and function is reported using data from IRB‐approved assessment and survey instruments and compared to data from students in virtual biochemistry courses without an activity. Unique course activities involving student movement can improve student learning of complex topics in biochemistry.

PDK4 Inhibition Ameliorates Melatonin Therapy by Modulating Cerebral Metabolism and Remyelination in an EAE Demyelinating Mouse Model of Multiple Sclerosis.

We recently showed that melatonin ameliorates the severity of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, efficiency of melatonin therapy was associated with side effects, manifested by slowing down of remyelination, through increasing the inhibitory effects of brain pyruvate dehydrogenase kinase-4 (PDK-4) on pyruvate dehydrogenase complex (PDC), a key enzyme in fatty acid (FA) synthesis during remyelination. In this study, we investigated the metabolic profile of FA synthesis using combination therapy of melatonin and diisopropylamine dichloroacetate (DADA), a PDK4 inhibitor, in EAE mice. Disease progression was monitored by recording the disability scores. Immunological, oligodendrogenesis and metabolic factors were also evaluated. Results showed that combination therapy of melatonin and DADA significantly reduced EAE disability scores, compared to melatonin, whereas DADA alone did not have any effect. In addition, co-therapy inhibited pro-inflammatory while increasing anti-inflammatory cytokines, significantly better than melatonin alone. Moreover, administration of combination drugs recovered the declined expression of oligodendrocytic markers in EAE, more potently than melatonin. Furthermore, co-therapy affected cerebral energy metabolism by significantly reducing lactate levels while increasing N-acetylaspartate (NAA) and 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase (HMGCR) levels. Finally, while melatonin increased lactate and PDK4 expression levels and greatly reduced PDC activity, co-therapy significantly restored PDC function while reducing the lactate levels. In summary, administration of melatonin with DADA increased the efficiency of melatonin treatment by eliminating the inhibitory effects of PDK4 on PDC’s function, a critical step for proper FA synthesis during remyelination.

Metabolic Dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Not Due to Anti-mitochondrial Antibodies.

Metabolic profiling studies have recently indicated dysfunctional mitochondria in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). This includes an impaired function of pyruvate dehydrogenase complex (PDC), possibly driven by serum factor(s), which leads to inadequate adenosine triphosphate generation and excessive lactate accumulation. A reminiscent energy blockade is likely to occur in primary biliary cholangitis (PBC), caused by anti-PDC autoantibodies, as recently proposed. PBC is associated with fatigue and post-exertional malaise, also signifying ME/CFS. We herein have investigated whether ME/CFS patients have autoreactive antibodies that could interfere with mitochondrial function. We found that only 1 of 161 examined ME/CFS patients was positive for anti-PDC, while all PBC patients (15/15) presented significant IgM, IgG, and IgA anti-PDC reactivity, as previously shown. None of fibromyalgia patients (0/14), multiple sclerosis patients (0/29), and healthy blood donors (0/44) controls showed reactivities. Anti-mitochondrial autoantibodies (inner and outer membrane) were negative in ME/CFS cohort. Anti-cardiolipin antibody levels in patients did not differ significantly from healthy blood donors. In conclusion, the impaired mitochondrial/metabolic dysfunction, observed in ME/CFS, cannot be explained by presence of circulating autoantibodies against the tested mitochondrial epitopes.

Pilot Study of Peripheral Muscle Function in Primary Biliary Cirrhosis: Potential Implications for Fatigue Pathogenesis

Primary biliary cirrhosis (PBC) is characterized in 95% of patients by autoantibody responses directed against the mitochondrial antigen pyruvate dehydrogenase complex (PDC). Although anti-PDC inhibits PDC function in vitro, mitochondrial function in vivo in PBC has not been examined. (31)P magnetic resonance spectroscopy was performed in PBC patients (n = 15) and fatigued (chronic fatigue syndrome/myalgic encephalomyelitis, n = 8), cholestatic (primary sclerosing cholangitis [PSC], n = 4), and normal (n = 8) controls to define mitochondrial function and pH regulation in peripheral muscle during exercise at 25% and 35% of maximum voluntary contraction. Normal, chronic fatigue syndrome/myalgic encephalomyelitis, and PSC subjects all showed close correlation between kinetics of adenosine diphosphate (ADP) and phosphocreatine (PCr) recovery after low-impact exercise, reflecting the normal tight regulation of PCr "response" by mitochondria to ADP "drive." This relationship was lost in PBC patients, indicating mitochondrial dysfunction (normal r(2) = 0.78, P < .005; PBC r(2) = 0.007, P = ns). Ratio between PCr and ADP recovery half-times (constant in controls, indicating normal mitochondrial responsivity) was significantly elevated in PBC patients (but not PSC) and was associated with anti-PDC levels. At higher levels of exercise PBC (but not PSC) patients showed excess muscle acidosis, with pH correlating with elevation of PCr/ADP recovery ratio, indicating a link to mitochondrial dysfunction. PBC patients alone also showed significant prolongation of muscle pH recovery time after exercise (unrelated to mitochondrial function), which correlated with clinical fatigue. PBC patients exhibit a variable degree of muscle mitochondrial dysfunction that manifests as excess acidosis after exercise. The extent to which patients can recover rapidly from acidosis appears to determine whether they are clinically fatigued.

The Dihydrolipoamide S-Acetyltransferase Subunit of the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.

Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases.

Optimal conditions for rapid and efficient reconstitution of pyruvate dehydrogenase complex (PDC) activity are demonstrated by using an improved method for the dissociation of the multienzyme complex into its constituent E1 (substrate-specific 2-oxoacid decarboxylase) and E3 (dihydrolipoamide dehydrogenase) components and isolated E2/X (where E2 is dihydrolipoamide acyltransferase) core assembly. Selective cleavage of the protein X component of the purified E2/X core with the proteinase arg C decreases the activity of the reconstituted complex to residual levels (i.e. 8-12%); however, significant recovery of reconstitution is achieved on addition of a large excess (i.e. 50-fold) of parent E3. N-terminal sequence analysis of the truncated 35,000-M(r) protein X fragment locates the site of cleavage by arg C at the extreme N-terminal boundary of a putative E3-binding domain and corresponds to the release of a 15,000-M(r) N-terminal fragment comprising both the lipoyl and linker sequences. In native PDC this region of protein X is shown to be partly protected from proteolytic attack by the presence of E3. Recovery of complex activity in the presence of excess E3 after arg C treatment is thought to result from low-affinity interactions with the partly disrupted subunit-binding domain on X and/or the intact analogous subunit binding domain on E2. Contrasting recoveries for arg C-modified E2/X/E1 core, and untreated E2/E1 core of the 2-oxoglutarate dehydrogenase complex, reconstituted with excess bovine heart E3, pig heart E3 or yeast E3 point to subtle differences in subunit interactions with heterologous E3s and offer an explanation for the inability of previous investigators to achieve restoration of PDC function after selective proteolysis of the protein X component.

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD+ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl2, 3.0 mM cysteine-HCl, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHCO3 mM ATP, 10 mM MgCl2, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sulphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis

In primary biliary cirrhosis (PBC), autoantibodies are produced to the M2 group of mitochondrial antigens, of which a major constituent is the E2 subunit of the pyruvate dehydrogenase complex (PDC). These antibodies, in addition to conventional reactivities with PDC, characteristically inhibit the catalytic function of PDC in vitro, as judged by a macroinhibition assay based on spectrophotometry. We describe a microinhibition assay adapted for microtitre plates and for an automated readout of results by an ELISA plate reader. We show that this microassay has a sensitivity similar to that of the macroassay. In a study of 83 sera, from PBC and other diseases, the inhibitory microassay proved specific for PBC. This automated inhibitory microassay for PBC-sera could become a primary laboratory procedure for the diagnosis of PBC, particularly because it may identify antibodies to the actual autoepitope on the PDC-E2.