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Metabolic Research Program, Research Institute, Hospital for Sick Children (M.C.M., N.M., V.L., J.A., B.H.R., J.M.C.), Toronto, Ontario, Canada M5G 1X8; Departments of Biochemistry and Pediatrics, University of Toronto (B.H.R.), Toronto, Ontario, Canada M5S 1A8; Universitats-Kinderspital beider Basel (E.R.B.), Postach, CH-4005 Basel, Switzerland; and Division of Metabolism and Molecular Pediatrics, University Children’s Hospital (M.R.B.), Postfach, CH-8032 Zurich, Switzerland
Address all correspondence and requests for reprints to: Dr. Brian H. Robinson, Metabolic Research Program, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail: bhr{at}sickkids.ca.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency.
Patients: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium.
Results: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient’s fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type).
Conclusion: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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2ß2 tetramer; dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The complex plays a pivotal role in metabolism, limiting the rate of oxidative glucose consumption, and is highly regulated to respond to all metabolic requirements. Several cofactors as well as reversible phosphorylation are required for this regulated mechanism (1, 2, 3).
The E1 2ß2 tetramer is the main site of regulation for the whole complex (4). Three serine residues located on E1 may be phosphorylated, with the consequent inhibition of complex activity (5, 6, 7, 8, 9). The phosphorylation is catalyzed by PDH kinase (PDK), of which there are four distinct forms (10, 11). Dephosphorylation to restore activity is catalyzed by pyruvate dehydrogenase phosphatase (PDP), which has two isoforms (12). The PDKs are regulated differentially by pyruvate as well as the ADP/ATP, nicotinamide adenine dinucleotide+ (NAD+)/reduced NAD+ (NADH+), and CoA/acetyl-CoA ratios, as reviewed by Roche et al. (13). High concentrations of pyruvate and the analog dichloroacetate (DCA) will inhibit PDK, decreasing the proportion of phospho-PDHc in the presence of PDP.
PDP is a serine/threonine phosphatase that is a member of the protein phosphatase 2C (PP2C) family (14). Mammalian PDP is a heterodimer, consisting of a regulatory and a catalytic subunit. Two isoforms of PDPc have been identified, PDP1 and PDP2 (12). Both PDP1 and PDP2 are magnesium dependent, although PDP2 requires a 10-fold higher Mg2+ concentration for activity. PDP1 requires both calcium and interaction with the E2 component of PDHc for optimal phosphatase activity (15, 16). The activity of the PDP2 isoform differs, in that it is not regulated by calcium, nor does it require the presence of E2 for activity. Both phosphatases are differentially expressed, with highest levels of PDP1 in skeletal muscle and highest levels of PDP2 in liver and adipocytes (12).
Deficiencies in PDHc activity are largely a result of defects in the X-linked PDHA1 gene, which encodes the E1 subunit (17, 18, 19), although rare mutations have been identified in the genes encoding E1ß (20), E2 dihydrolipoamide acetyltransferase, E3 dihydrolipoamide dehydrogenase, and E3-binding protein (21, 22). PDHc deficiency is usually diagnosed by measurement of PDHc activity in fibroblasts or lymphocytes, measuring the enzyme in the native state. The activity of the enzyme complex can also be measured after the activation of PDHc, either in the presence of Ca2+ and Mg2+ (23) or after preincubation of the cells with DCA (24). We have performed these tests with patient fibroblasts to diagnose two male siblings with deficiency of native, but not activated, PDHc. The neonatal presentation of the brothers included hypotonia, feeding difficulties, elevated lactate levels, and delayed psychomotor development. We have shown that this failure of activation is due to mutation of the PDP1 gene.
Defects in the activation of PDHc by PDPs have been proposed as an inborn error contributing to lactic acidemia on several occasions, but the genetic and biochemical coordinate proof was lacking (25, 26, 27, 28, 29, 30). For the first time, we define a genetic defect of PDP.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Patients 1 and 2 are two male siblings of Turkish origin, whose parents are first cousins. They both presented neonatally with hypotonia, feeding difficulties, and elevated lactate. Currently, the brothers are 12 and 10 yr old and are surviving well on a ketogenic diet that is not strictly maintained. Plasma lactate is approximately 3 mmol at complete rest and quickly rises to 9–10 mmol with minimal exercise. Both have slightly delayed psychomotor development.
Cultured skin fibroblasts were grown from forearm skin biopsies (taken with informed consent) in MEM culture medium (11 mM glucose) and 10% fetal calf serum (Wisent, Inc., Saint-Jean-Baptiste de Rouville, Quebec, Canada). Lymphocytes were isolated from blood samples taken from both patients (with informed consent) by the Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) method (31). The cell pellet is resuspended in 1 ml RPMI 1640 culture medium with 20% fetal calf serum, 2 µg cyclosporin, and 500 µl filtered supernatant from B95-8 cells (Epstein-Barr virus-producing marmoset lymphoblasts from Coriell Cell Repositories, Camden, NJ). The cells are incubated for 1 month at 37 C with 5% CO2, with biweekly exchange of culture medium. After 1 month, the transformed lymphoblasts were grown in RPMI 1640 culture medium and 10% fetal calf serum.
Molecular genetics techniques
RNA was isolated from cultured skin fibroblasts using TRIzol (Invitrogen Life Technologies, Inc., Carlsbad, CA). Genomic DNA was isolated using Puregene genomic DNA isolation kit (Versagene, Minneapolis, MN). Full-length PDP1 cDNA sequence was amplified by RT-PCR using SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc.) and Platinum Hi-Fi Taq polymerase (Invitrogen Life Technologies, Inc.). The following oligonucleotide primers were used for PCR: PDP1 forward, 5'-GCCTGCCACTGTTCTCTGAT-3'; PDP1 reverse, 5'-GAATTGCCAGTAAAGAGCCACT-3' (for amplification of the entire coding region); and PDP1int forward, 5'-TGCCTTCAAGAGGCTTGATAA-3'; PDP1int reverse, 5'-AATGCCCTAAATGGCATCAG-3' (for amplification of the region flanking the patient deletion). Mature forms (m) of PDP1 and PDP2 for protein expression were amplified from full-length cDNA with the following primers: mPDP1 forward, 5'-CATATGTATGCTTCCACACCACAGAAAT-3'; mPDP1 reverse, 5'-CTCGAGGCCAGTAAAGAGCCACTCAC-3'; mPDP2 forward, 5'-CATATGTCAACAGAGGAAGATGAT-3'; and mPDP2 reverse, 5'-CTCGAGTAGGATG- GGAGATTCTTA-3'.
RT-PCR products were cloned into pCR2.1 vector (TOPO TA cloning kit, Invitrogen Life Technologies, Inc.), pTZ75R/T vector (InsTAclone PCR product cloning kit, MBI Fermentas, Hanover, MD), or pET-28 vectors (Novagen, Madison, WI). All clones were sequenced by fluorescent sequencing methods (Center for Applied Genomics, Hospital for Sick Children). BslI restriction enzyme digests (New England Biolabs, Beverly, MA) were performed with PCR products amplified from genomic DNA.
Immunoblotting
For Western blot analysis, mitochondria were prepared from fibroblasts (32) and lymphoblasts and were resolved through a 12.5% separating SDS-PAGE gel. Lymphoblast mitochondria were prepared from 20 ml lymphoblasts, which were pelleted by centrifuging at 375 x g for 5 min in an Eppendorf 5804 benchtop centrifuge (Brinkmann Instruments, Inc., Westbury, NY). The pellet was washed three times with medium A [300 mM sucrose, 1 mM EGTA, 20 mM 4-morpholinepropanesulfonic acid (MOPS; pH 7.4), and 1 g/liter BSA] and then resuspended in 1 ml medium A containing 10 mM triethylamine and 0.1 mg/ml digitonin (medium B). The suspension was incubated on ice for 3 min. Two milliliters of medium C were added [500 mM sucrose, 20 mM MOPS (pH 7.4), 1 mM EDTA] and homogenized for 30 strokes using a Teflon homogenizer (Wheaton, Millville NJ). This was then centrifuged at 16,100 x g for 5 min in an Eppendorf 5415D benchtop centrifuge. The supernatant was removed to a fresh Eppendorf tube and centrifuged at 16,100 x g for 5 min at 4 C. The mitochondrial pellet was resuspended in 100 µl medium C.
The SDS-PAGE gel was electroblotted onto nitrocellulose membrane and blocked with 5% skim milk in Blotto (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The gel was then probed with rabbit antibovine heart total PDHc, antihuman PDP1, or antihuman citrate synthase antibodies in 3% BSA/Tris-buffered saline with Tween 20. Immunoreactive proteins were visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Wellesley, MA).
Recombinant enzymes
The four isoforms of mature human PDKs were cloned and purified as previously described (33). The mitochondrial precursors of PDP, isoforms 1 and 2, and isoform 1 containing the mutation (PDP1m), were cloned from cDNA derived from human fibroblasts as described above. The mature forms of PDP1, PDP2, and PDP1m were amplified from their respective clones and ligated into pCR2.1-TOPO vector (Invitrogen Life Technologies, Inc.). Each isoform was subcloned into pET-28 expression vectors (Novagen) with the histidine tag at the N terminus. The coding regions were then verified by sequence analysis. The resulting plasmids were cotransformed into Escherichia coli BL21[DE3] cells (Novagen) with a plasmid containing the chaperone GroEL/GroES (gift from Dr. Anthony Guttenbery, DuPont, Wilmington, DE). The cells were grown to an OD550 of 0.5 at 37 C. Protein expression was induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactoside and was expressed for 60 h at 15 C. The phosphatases containing the N-terminal six-histamine tag were purified by first binding the protein to nickel resin in 20 mM sodium phosphate buffer, pH 7.8, containing 5 mM imidazole and 500 mM NaCl, then washing the protein-bound resin with 20 mM sodium phosphate buffer, pH 7.3, containing 50 mM imidazole and 500 mM NaCl. The phosphatases were eluted from the resin with sodium phosphate buffer, pH 7.3, containing 200 mM imidazole and 500 mM NaCl. Isoforms 1 and 2 were purified to greater than 75% purity, and PDP1m was purified to greater than 60% purity, as determined by electrophoresing the purified proteins through SDS-PAGE gels and staining with Coomassie Blue. The proteins were stored in 30 mM MOPS buffer, pH 7.4, with 5% glycerol and 1 mM dithiothreitol (DTT).
PDP1 activity measurements (spectrophotometric assays)
Purified porcine PDHc was phosphorylated by incubation of the complex with 10 mM ATP, 50 mM Tris-HCl (pH 7.5), 340 mM sucrose, 100 mM KCl, 1 mM EDTA, 10 mM MgCl2, 20 mM sodium fluoride, 6 µg oligomycin, 1 µl protease inhibitor cocktail (Calbiochem, La Jolla, CA), and 10 µg of each isoform of PDK for 15 min at 30 C. The residual ATP and fluoride were removed by ultracentrifugation at 100,000 x g in a TL-100 centrifuge (Beckman Coulter, Fullerton, CA) for 90 min at 4 C. The phosphorylated PDH was resuspended in assay buffer containing 100 mM MOPS (pH 7.4), 1 mM DTT, 10 mM MgCl2, and 100 µM thiamine pyrophosphate (TPP). The generation of NADH through the activity of 40 mU phosphorylated PDH was followed spectrophotometrically at 340 nm and 30 C in a final volume of 1 ml. The reaction was initiated by the addition of substrates CoA (250 µM), pyruvate (2.5 mM), and NAD+ (5 mM).
The abilities of PDP1, PDP2, and PDP1m to reactivate the phosphorylated PDH were investigated. Various amounts of each recombinant phosphatase were added to the final reaction mixture and incubated for 2 min at 30 C before the addition of substrate. Assays for PDP1 and PDP1m contained 2 mM calcium. All spectrophotometric assays were performed on a UV/vis instrument (model UV 300, Cary, Varian, Inc., Palo \E \E Alto, CA). Each assay was performed in duplicate.
PDP1 activity measurements (14CO2 release assay)
Immediately after harvesting control and patient fibroblast cultures, the cells were resuspended in buffer to approximately 2 mg/ml protein. The buffer contained 100 mM MOPS (pH 7.4), 2 mg/ml BSA, 10 mM MgCl2, 100 µM TPP, 2 mM calcium, and 0.05% Triton X-100. Various amounts of phosphatase proteins were added to the cell lysates and incubated at 30 C for 10 min with occasional stirring. The cell lysates were then snap-frozen in liquid nitrogen for measurements of native PDHc activity in the absence of added DCA according to the protocol described below. Each assay was performed in duplicate.
PDHc activity assay
PDHc activity was assayed according to the method of Sheu et al. (24) with the following adaptations. Immediately after harvesting fibroblast cultures, the cells were resuspended to approximately 2 mg/ml protein in PBS with or without pretreatment with 5 mM DCA to assay either native activity (in the absence of DCA) or DCA-activated PDHc activity. Both native and DCA-activated cells were incubated for 10 min at 37 C in a shaking water bath; stopped with a one third cell extract volume of a stopping solution containing 40% (vol/vol) ethanol, 25 mM sodium fluoride, 25 mM EDTA, and 0.0001% ß-mercaptoethanol (pH 7.4); then snap-frozen in liquid nitrogen. The treated cells were thawed, and 50 µl were added to 200 µl incubation mix contained in 10-ml Erlenmeyer flasks. The radiolabeled CO2 was collected in 0.2 ml benzethoniom hydroxide that was held in wells attached to rubber stoppers, which were used to cap the flasks. The incubation mix contained 100 mM potassium phosphate buffer (pH 7.4), 10 mg/ml fatty acid-free BSA, 1 mM DTT, 1.5 mM NAD+, 3 mM MgCl2, 10 mM EDTA, 125 µM TPP, 0.2 mg/ml CoA, 0.1 mg/ml sodium sulfite, and 0.106 mM pyruvate with 0.25 µCi/ml [1-14C]pyruvate (Amersham Biosciences, Arlington Heights, IL). The reaction was incubated for 10 min in a 37 C shaking water bath. The reaction was stopped with 100 µl 10% trichloroacetic acid (TCA), and CO2 was collected for 1 h. The radioactive CO2 associated with benzethoniom hydroxide was placed in 5 ml toluene scintillation fluid and counted on a Beckman LS 6500 scintillation counter. Each assay was performed in duplicate.
[2-14C]Pyruvate whole cell oxidation assay
Whole cell assays with [2-14C]pyruvate (PerkinElmer) as substrate follow the elution of CO2, through the activity of -ketoglutarate dehydrogenase, during the second round of the citric acid cycle. These assays were performed by a modification of the method described by Robinson et al. (34). Using the same experimental setup as that for the PDHc activity assay above, 50 µl cell suspension were added to 200 µl reaction mixture (PBS with 205 µM cold pyruvic acid and 0.737 µCi/ml [2-14C]pyruvate) and incubated for 1 h at 37 C. After stopping the reaction, CO2 was collected for 1 h and counted as described above. Each assay was performed in triplicate.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In cultured cells, PDHc activity is typically assessed by measuring the rate of decarboxylation of 14CO2 from [1-14C]pyruvate in the presence of cofactors. Two measurements are made: one of the native activity, representing the proportion of PDHc in the active, dephosphorylated state, and the other after full activation of the complex in the presence of DCA. In fibroblasts, patient 1 showed 30% PDHc native activity of control values, although DCA-activated PDHc was 77% of control values. Patient 2 had 32% native activity, with normal activity upon DCA activation (Table 1
). In lymphoblasts, patient 1 had 40% native activity and normal DCA activity, whereas patient 2 had 37% native activity of control values, and DCA-activated PDHc was 67% of control values (Table 1). In the presence of DCA, the proportion of phosphorylated PDHc was decreased through inhibition of associated kinases, and the patient’s PDHc activity began to approach control levels. These results are contrary to typical cases of PDHc deficiency in which the defect in one of the complex catalytic subunits, such as E1, results in low native and DCA-activated enzyme rates.
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The entire coding sequence for PDP1 (NM_018444), PDP2 (NM_020786), and PDPR (NM_017990; the gene encoding the regulatory subunit of PDP) were amplified by RT-PCR from cDNA prepared from both patients and control fibroblasts and ligated into cloning vectors. Three clones of each gene for both brothers and one from control fibroblasts were then sequenced. A 3-bp deletion was identified in all cDNA clones derived from the two brothers (Fig. 1A). Genomic DNA was prepared from both patient and control fibroblasts and lymphoblasts, and a 345-bp PCR product containing the deletion region was amplified. The products were digested with restriction endonuclease BslI. Control DNA PCR product was digested into three products of 203, 133, and 9 bp. Mutant DNA PCR product was digested into only two products of 333 and 9 bp (Fig. 2). Thus, the mutation was identified as homozygous in both brothers and was expressed from both fibroblasts and lymphoblasts. The 3-bp deletion resulted in the deletion of leucine 213 of the mature protein (Fig. 1B). This amino acid was highly conserved through evolution from humans to yeast, although not in the distantly related PP2C (Fig. 1C). No mutations in PDP2 or PDPR were found (data not shown).
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. A much lower amount of PDP1 protein compared with the control value was observed for both patients. Relative to citrate synthase, PDP1 levels appeared to be about 20% of the control value, suggesting lower import or stability. All subunits of PDHc appeared to be present at normal levels. Patients with a defect in E1 or other subunits of the complex usually show reduced protein levels of that subunit, due to degradation of the mutant protein. These results suggest that the mutation of PDP1 is not affecting the assembly of PDHc.
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The cDNAs of mature PDP1, wild-type (wt) and mutant, and PDP2 were ligated into a protein expression vector, expressed, and purified. The activities of these recombinant proteins were determined spectrophotometrically using purified PDHc. Phosphatase activity was measured as the initial rate of reactivation of phospho-PDHc. Phospho-PDHc was generated upon incubation of purified PDHc with purified PDKs and ATP. This fully phosphorylated complex exhibited very low residual activity. Increasing amounts of the recombinant phosphatase proteins were incubated with the phosphorylated PDH complex. The initial velocity of the reactivated PDH complex was plotted against the amount of phosphatase added. As shown in Fig. 4A, the initial velocity of PDHc activity increased as the amount of phosphatase was increased. Both wild-type and mutant PDP1 required 2 mM calcium for optimal phosphorylase activity. Concentrations of calcium above 2 mM were inhibitory to PDHc activity under all conditions tested. The wt-PDP1 became inhibitory above 2 µg, with an apparent inhibitory constant (Ki) of 14 ± 2.9 µg. The apparent Michaelis-Menten constant (Kmapp) is 0.18 ± 0.05 µg for wt-PDP1 and 5.60 ± 2.04 µg for PDP2. These studies suggest that the efficiency of wt-PDP1 is 50 times greater for phospho-PDH complex than for PDP2. The activity of mutant PDP1 was too low to accurately determine the Kmapp. However, it was noticed that mutant PDP1 required up to 20-min incubation with phosphorylated PDHc for optimal activity (data not shown).
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If the mutation that deletes amino acid L213 from PDP1 causes PDHc deficiency, then it is predictable that normal complex activity could be restored upon the addition of recombinant protein to cell lysates. Fibroblast PDHc activities in the presence and absence of wt-PDP1 PDP1 were determined. Increasing amounts of wt PDP1 were added to both control and patient cell lysates. Figure 4B
shows the effects of increasing amounts of wt-PDP1 on PDHc activity. Although control fibroblast PDHc activity increased only slightly, full PDHc activity was restored in patient fibroblasts upon the addition of 2 µg wt protein. In the absence of calcium, 10 µg wt-PDP1 failed to restore patient PDHc activity, indicating that, similar to native PDP1, the recombinant protein has a calcium requirement for phosphatase activity.
Mechanism of mutant PDP1 inhibition of PDHc activity
To better understand the structural significance of the leucine residue deletion found in these patients, we modeled the amino acid sequence of PDP1 into the structure of the related Mn2+/Mg2+-dependent PP2C (12, 35) for which the three-dimensional (3D) structure has been solved (Protein Data Bank identification no. 1A6Q) (36). It is well documented that related proteins that catalyze similar reactions typically have very similar tertiary structures, and conserved residues are retained in 3D space. Conserved amino acid regions of PDP1 were threaded into the structure of PP2C with DeepView/SwisPdbViewer software (version 3.7, free software available at http://au.expasy.org). Two regions of the PDP1 amino acid sequence that are absent in PP2C were modeled as loops. The aspartic acid residues that are conserved among PDP1, PDP2, and PP2C are highlighted in Fig. 5. The position of L213 is inferred from the structural alignment of conserved regions and active site residues. Although L213 is distant from the active site, it is found at the beginning of ß-strand B6. An aspartic acid residue D220, which is critical for the orientation of three conserved active site aspartic acid residues, D73, D347, and D446, is shown at the end of ß-strand B6. If the leucine residue is removed, the position of D220 will be altered and may no longer form the hydrogen bonds that may be responsible for the correct orientation of the aspartic acid residues and therefore critical for phosphatase activity. The distance between these aspartic acid residues allowing for hydrogen bonding as well as the positions of phosphate and manganese ions are the same as those found for the structure of PP2C. The Mn2+ ions coordinating directly with aspartic acid residues may stabilize the active site for partial activity of the mutant protein.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A 3-bp deletion was identified in PDP1, which results in the loss of amino acid L-213 from the mature protein sequence. The cDNA of mutant PDP1 could be successfully amplified by RT-PCR, indicating that the mRNA transcript was present. The mutation was shown to be homozygous in the patients in both fibroblasts and lymphoblasts, demonstrating that wt-PDP1 protein was not expressed in these tissues. Using a PDP1 antibody, the mutant protein could be detected on a Western blot, but was present in very low amounts compared with the control. No immunoreactive degradation products were detected. To verify that deletion of L-213 in PDP1 gives rise to the clinical phenotype of our patients, we performed PDHc assays with patient cell lysates in the presence of purified recombinant wt-PDP1. We were able to restore full PDHc activity in the patients’ fibroblasts.
The crystal structure of PDP1 has not been determined, but homology can be inferred from cytosolic Mn2+/Mg2+-dependent PP2C. A model for PDP1 based on this 3D structure has been proposed previously (12). The basic structure of PP2C is a ß-sandwich, surrounded by -helices. The catalytic core contains a binuclear metal-binding site that lies at the upper cleft between the two ß-sheet structures that form the ß-sandwich. The binuclear metals associate with a phosphate ion, representing the phospho-amino acid of the target protein, through coordinated water molecules at the active site. Based on amino acid homology between conserved familial regions of PDP1 and PP2C, the residues for PDP1 were threaded into the structure of PP2C. The essential metal-coordinating aspartic acid residues are invariant among proteins of the PP2C family. The importance of invariant aspartic acid residues D347 and D445 to the activity of PDP1 has recently been identified by site-directed mutation (37). Our model suggests that residue D220 is required for the correct orientations of D73, D347, and D445. D220 is located at the C terminus of ß-strand 6. L213, which is lost in our two patients, is located at the N terminus of ß-strand 6. Therefore, the loss of L213 would shift the position of D220 and may result in a disruption of the hydrogen bonding network of the active site. This hydrogen bonding network is necessary for the formation of a binuclear metal center, which is required for interaction through coordinated water molecules to the phosphate group covalently linked to one of three serine residues of the E1-subunit. Thus, this mutation may reduce the affinity of the protein for metals, resulting in a decrease in phosphatase activity, as shown by biochemical experimentation. Based on homology to the structure of PP2C, the deletion of L213 may also cause a disruption of the bridging atop the two ß-sheets that form the ß-sandwich active site core. This disruption could result in protein misfolding, either before reaching the mitochondria or after import, and subsequent rapid degradation of PDP1, as suggested by Western blot analysis.
The two patients therefore have a defect in PDP1 that will result in disrupted regulation of the PDHc. Activation of the complex using DCA, which acts by inhibiting PDK, resulted in nearly normal activated complex rates. Although the activity of recombinant PDP2 is less than 50 times that of recombinant PDP1, it appears that endogenous PDP2 in the patients’ fibroblasts, under conditions of DCA activation, is able to successfully dephosphorylate the complex, resulting in normal activated PDHc activity.
In summary, we have determined unequivocally that a genetic basis for PDP deficiency exists. From our initial description of a patient with this defect in 1975 (27), we can now, 29 yr later, describe the molecular basis.
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Footnotes |
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First Published Online April 26, 2005
Abbreviations: CoA, Coenzyme A; 3D, three-dimensional; DCA, dichloroacetate; DTT, dithiothreitol; m, mature; MOPS, 4-morpholinepropanesulfonic acid; NAD, nicotinamide adenine dinucleotide; NADH, reduced NAD; PDH, pyruvate dehydrogenase; PDHc, PDH complex; PDK, PDH kinase; PDP, PDH phosphatase; PDPc, PDP catalytic subunit; PDPR, PDP regulatory subunit; PP2C, protein phosphatase 2C; TCA, trichloroacetic acid; TPP, thiamine pyrophosphate; wt, wild type.
Received January 20, 2005.
Accepted April 19, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-Keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci USA 62:234–241-Keto acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria. Proc Natl Acad Sci USA 64:227–234 subunit of the human pyruvate dehydrogenase complex. Genomics 4:174–181[CrossRef][Medline]
subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency. Hum Mutat 15:209–219[CrossRef][Medline]
-Keto acid dehydrogenase complexes. Basel, Switzerland: Birkhäuser; 249–269
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), PDP2 (
), and mutant PDP1 (
). The data are an average of two experiments and were fitted with PRISM software (GraphPad, San Diego, CA). B, Effects of wt and mutant PDP1 on PDHc activity in fibroblast lysates. The activities of patient and control PDHc were determined as described in Patients and Methods. The effects of increasing amounts of wt-PDP1 on control and patient PDHc activities are plotted. 
, PDHc activity in the absence of calcium. Each value is the SD of two experiments performed in duplicate.