E2 Subunit Of Pyruvate Dehydrogenase Research Articles

Externalization of Mitochondrial PDCE2 on Irradiated Endothelium as a Target for Radiation-Guided Drug Delivery and Precision Thrombosis of Pathological Vasculature.

Endothelial cells are highly sensitive to ionizing radiation, and exposure leads to multiple adaptive changes. Remarkably, part of this response is the translocation of normally intracellular proteins to the cell surface. It is unclear whether this ectopic expression has a protective or deleterious function, but, regardless, these surface-exposed proteins may provide unique discriminatory targets for radiation-guided drug delivery to vascular malformations or tumor vasculature. We investigated the ability of an antibody–thrombin conjugate targeting mitochondrial PDCE2 (E2 subunit of pyruvate dehydrogenase) to induce precision thrombosis on irradiated endothelial cells in a parallel-plate flow system. Click-chemistry was used to create antibody–thrombin conjugates targeting PDCE2 as the vascular targeting agent (VTA). VTAs were injected into the parallel-plate flow system with whole human blood circulating over irradiated cells. The efficacy and specificity of fibrin-thrombus formation was assessed relative to non-irradiated controls. The PDCE2-targeting VTA dose-dependently increased thrombus formation: minimal thrombosis was induced in response to 5 Gy radiation; doses of 15 and 25 Gy induced significant thrombosis with equivalent efficacy. Negligible VTA binding or thrombosis was demonstrated in the absence of radiation or with non-targeted thrombin. PDCE2 represents a unique discriminatory target for radiation-guided drug delivery and precision thrombosis in pathological vasculature.

Immune pathogenesis of primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune liver disease, mainly characterized by chronic progressive cholestasis. The root cause of PBC is the loss of immune tolerance to autoantigen E2 subunit of pyruvate dehydrogenase (PDC-E2). The unique immunobiological characteristics of intrahepatic bile duct epithelial cells make it an active participant in the pathogenesis of PBC. In recent years, the detection rate of PBC has been increasing year by year, but the clinical situation of ursodeoxycholic acid monotherapy has not changed. Therefore, an in-depth understanding of the immune pathogenesis of PBC will help clinicians better prevent and treat diseases.

Nitric Oxide Mediates Direct Restriction of Pyruvate Dehydrogenase Complex via Generation of Nitroxyl During Macrophage Polarization

M1 macrophages “commit” to a metabolic state where increased aerobic glycolysis sustains fast ATP production, independently of Oxidative Phosphorylation (OXPHOS). This is associated with a “broken” mitochondrial TCA cycle and accumulation of metabolites like citrate, succinate and itaconate, important for cellular functions. We have shown that induced Nitric Oxide (NO) levels in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice activated with lipopolysaccharide and interferon gamma (LPS + IFNγ) are necessary and sufficient for the repression of OXPHOS and for metabolic reprogramming at mitochondrial Aconitase (ACO2) and Pyruvate Dehydrogenase (PDH). We hypothesize that NOdirectly targets the PDH complex via its critical cofactor lipoate, interacting with its thiol motifs. High-sensitivity proteomics on PDH complex shows that stimulated BMDM and cells exposed to NO have stalled enzymatic machinery, and this correlates with NO-associated lipoate on E2 subunit of PDH. Moreover, through native in gel activity assays we found decreased enzymatic activity of PDH-E3 and shifts in molecular weight of both PDH-E3 and E3 binding protein (E3Bp); in addition we found these positive for nitrogen specie-mediated modifications, which were not easily reversible with common reducing agents. Using chemical approaches we show that interaction of lipoate with NO can generate a reduced NO radical, nitroxyl (HNO), which can interact with thiols to form unstable intermediates that spontaneously rearrange to a sulfinamide; this represents a nearly irreversible thiol modification. Consistently, mass spectrometry analysis of recombinant protein reveals HNO-dependent post translational modification (PTM) of PDHE3. Structurally, HNO-specific modifications on E3 strongly impair homodimer formation, essential for activity. Our work is the first to demonstrate that HNO is produced physiologically in proinflammatory macrophages, and it is locally released within the interaction with lipoate moiety at the level of PDH-E2 and E3Bp, facilitating irreversible modification of distal E3. In turn this leads to inability for deacetylation of lipoate and global impairment of enzyme function. Finally, since the same E3 subunit is shared with two other key cellular metabolic enzymes, Oxoglutarate and Branched-chain alpha-keto acid Dehydrogenases (OGDH and BCKDH) we predict that NO could be responsible for orchestrating macrophage energy metabolism during inflammation also by limiting the activity of these dehydrogenases through irreversible modifications, opening the possibility for new therapeutic manipulations.

Similar clinical outcome of AMA immunoblot-M2-negative compared to immunoblot-positive subjects over six years of follow-up

ABSTRACT Background: The detection of anti-mitochondrial antibodies (AMA) is considered a hallmark in diagnosing primary biliary cholangitis (PBC). The most important AMA-subtype is AMA-M2 directed against the E2-subunit of pyruvate dehydrogenase. It is common clinical interpretation that lack of M2 due to immunoblotting (IB) indicates absence of specific auto-reactivity. We aimed to define whether M2-IB confirmation is linked to clinical outcomes. Methods: Our cohort comprised 302 patients who tested positive for AMA with indirect immunofluorescence between 2006 and 2015. One hundred and eighty-four subjects (60.9%; male n = 29 [15.8%]) were tested M2-positive by confirmatory IB, whereas 118 subjects were IB-M2-negative (39.1%; male n = 25 [21.2%]). The natural history of 236 patients (78.1%) was evaluated by clinical follow-up (FU) assessing causes of death, leading health condition and response to PBC standard therapy if applicable. Results: Mean time to FU was 6.8 years. Twenty-eight M2-positive patients (15.2% of 184) and 28 M2-negative patients (23.7% of 118) had died (p = 0.0958). Thirty-four M2-positives (18.5%) and 32 M2-negatives (27.1%) were not available for FU. According to the clinical course by the time of FU, subjects were allocated to one of four groups: a) 34 patients had known PBC with n = 16 having an adequate and 18 an inadequate treatment response, b) 1 de novo PBC was detected, c) 13 were AMA-positive without biochemical evidence of PBC and d) 9 subjects were tested AMA-negative at FU. These numbers were comparable to M2-positive subjects with similar long-term clinical outcome. Conclusion: Our data suggest that the clinical value of confirmatory M2 immunoblotting in the diagnostic routine of PBC is overestimated as the clinical course appears not to be related to the test result.

Solanum lycopersicum (tomato) possesses mitochondrial and plastidial lipoyl synthases capable of increasing lipoylation levels when expressed in bacteria

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.

Deterioration of mitochondrial bioenergetics and ultrastructure impairment in skeletal muscle of a transgenic minipig model in the early stages of Huntington's disease.

ABSTRACTSkeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model.This article has an associated First Person interview with the first author of the paper.

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62–66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.

Overproduction of α-Lipoic Acid by Gene Manipulated Escherichia coli.

Alpha-lipoic acid (LA) is an important enzyme cofactor widely used by organisms and is also a natural antioxidant for the treatment of pathologies driven by low levels of endogenous antioxidants. In order to establish a safer and more efficient process for LA production, we developed a new biological method for LA synthesis based on the emerging knowledge of lipoic acid biosynthesis. We first cloned the lipD gene, which encodes the lipoyl domain of the E2 subunit of pyruvate dehydrogenase, allowing high levels of LipD production. Plasmids containing genes for the biosynthesis of LA were subsequently constructed utilizing various vectors and promotors to produce high levels of LA. These plasmids were transformed into the Escherichia coli strain BL21. Octanoic acid (OA) was used as the substrate for LA synthesis. One transformant, YS61, which carried lipD, lplA, and lipA, produced LA at levels over 200-fold greater than the wild-type strain, showing that LA could be produced efficiently in E. coli using genetic engineering methods.

Abstract B08: Metabolic adaption in inflammatory macrophages through the modulation of Fe-S cluster biogenesis factors

Abstract Macrophages are among the most abundant normal cells in the tumor microenvironment, and the mechanistic overlap in the metabolic changes in glycolytic cancer cells and inflammatory immune cells suggest that insights into the mechanisms underlying the metabolic changes could be useful in the treatment of both cancers and inflammatory diseases. Metabolic choices in immune cells are tightly linked to cell fate and function. Inflammatory immune cells, such as M1 macrophages and T-helper 17 cells, undergo a shift from OXPHOS to enhanced glucose uptake, glycolysis and the pentose phosphate pathway, whereas anti-inflammatory cells, such as M2 macrophages and regulatory T cells have lower glycolytic rates and higher levels of oxidative metabolism. The metabolic switch from OXPHOS to aerobic glycolysis (Warburg effect) in Toll-like receptor (TLR)-stimulated myeloid cells has been shown to result from the activation of glycolysis through the action of AKT and HIF-1α signaling, with mitochondrial respiration passively decreasing due to NF-kB mediated induction of inducible nitric oxide synthase (iNOS), which produces the toxic gas nitric oxide that damages the Fe-S cluster cofactors that are essential for mitochondrial electron transport. The results reported here indicate that the pro-inflammatory activation of macrophages also involved the active suppression of mitochondrial pyruvate catabolism and respiration through the down-regulation of several genes in the Fe-S cluster biogenesis pathway. A decrease in Fe-S cluster biogenesis/repair not only resulted in a decrease in the activities of Fe-S cluster dependent enzymes including the respiratory complexes I and II, mitochondrial and cytosolic aconitases, and ferrochelatase, but also reduced lipoylation in the E2 subunit of pyruvate dehydrogenase (PDH) complex, thereby inhibiting pyruvate catabolism through the TCA cycle. Inhibition of glycolytic metabolism limited the TLR-stimulated repression of Fe-S cluster biogenesis factors, consistent with a vital survival function of Fe-S cluster biogenesis in cells that depend on mitochondrial ATP production. Activation of AMPK, inhibition of mTOR, and inhibition of Nf-kB all resulted in reduced TLR-induced suppression of Fe-S cluster biogenesis factors. These results suggested that the regulation of Fe-S cluster biogenesis factors are highly sensitive to cellular metabolic needs and are subject to the regulation of metabolic regulators AMPK and mTOR and inflammation modulator Nf-kB. Our results suggested that, in addition to iNOS induction, repression of Fe-S cluster biogenesis/repair may serve to suppress OXPHOS to promote the production of mitochondrial ROS for host defense, and to drive prolonged glycolytic metabolism to produce ATP, NADPH, and biosynthetic precursors to meet the metabolic and anabolic demands of host defense. Citation Format: Wing-Hang Tong, Nunziata Maio, Tracey A. Rouault. Metabolic adaption in inflammatory macrophages through the modulation of Fe-S cluster biogenesis factors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B08.

Abstract 15783: Reduction in Hepatic Tristetraprolin Increases Oxidative Metabolism and Protects Against High-Fat Diet Induced Diabetes

Introduction: Type II diabetes mellitus (T2DM) is a growing health problem affecting over 29 million Americans and individuals with T2DM have increased mortality after myocardial infarction and stroke. Thus, it is imperative to find novel treatments for diabetes to offset the increased risk of cardiovascular disease (CVD) related mortality. Tristetraprolin (TTP) is an mRNA binding protein first identified as an insulin responsive gene. It binds to AU-rich elements (AREs) in the 3’ untranslated region (UTR) of certain transcripts and promotes their degradation. Reduced TTP expression has been observed in human patients with obesity and insulin resistance, and computational analysis suggests that TTP may bind to and degrade the mRNA of key enzymes involved in glucose oxidation. Thus, we hypothesized that downregulation of TTP would increase glucose oxidation and protect against T2DM. Results: We found hepatic expression of TTP to be decreased in diabetic mice. Using an in silico analysis to identify mRNAs that are targeted by TTP and play a role in glucose metabolism, we identified the pyruvate dehydrogenase-E2 subunit (PDH-E2) to contain several conserved TTP binding sites in its 3’ UTR. PDH-E2 expression was significantly increased (mRNA > 1.4-fold; protein > 2-fold) in hepatocytes isolated from liver-specific TTP knockout (KO) mice. Furthermore, measurement of PDH-E2 mRNA stability showed that PDH-E2 mRNA is significantly stabilized with TTP deletion, indicating that TTP regulates PDH-E2 mRNA. We then assessed whether the regulation of PDH-E2 by TTP alters glucose metabolism. Using Seahorse, we found a 1.7-fold increase in oxidative metabolism in TTP KO cells fed with glucose and pyruvate. This increase was reversed with siRNA mediated downregulation of PDH-E2. Systemically, liver-specific TTP KO mice fed a high-fat diet had significantly lower blood glucose levels after glucose tolerance tests and insulin tolerance tests. Conclusion: Our results suggest that a decrease in TTP protects against the development of T2DM by increasing PDH-E2 expression and subsequent glucose oxidation in the liver. Together, these data provide a novel, potential therapeutic target for T2DM, a significant modifiable risk factor contributing to CVD mortality.

The structure of lipoyl synthase, a remarkable enzyme that performs the last step of an extraordinary biosynthetic pathway.

Lipoic acid is assembled on its cognate proteins (e.g. the E2 subunit of pyruvate dehydrogenase). An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthetase to a specific lysine residue of the cognate protein followed by sulfur insertion at C6 and C8 of the octanoyl chain. The challenging chemistry of this last step is performed by the radical S-adenosylmethionine (SAM) enzyme lipoyl synthase (LipA). In this issue of the Biochemical Journal, Harmer et al. report the first crystal structure of a lipoyl synthase and demonstrate that it contains two [4Fe-4S] clusters, the canonical radical SAM cluster plus a second auxiliary cluster having an unprecedented serine ligand. The structure provides strong support for the model in which the auxiliary cluster donates the lipoate sulfur atoms.

Engineered drug-protein nanoparticle complexes for folate receptor targeting

Nanomaterials that are used in therapeutic applications need a high degree of uniformity and functionality which can be difficult to attain. One strategy for fabrication is to utilize the biological precision afforded by recombinant synthesis. Through protein engineering, we have produced ∼27-nm dodecahedral protein nanoparticles using the thermostable E2 subunit of pyruvate dehydrogenase as a scaffold and added optical imaging, drug delivery, and tumor targeting capabilities. Cysteines in the internal cavity of the engineered caged protein scaffold (E2 variant D381C) were conjugated with maleimide-bearing Alexa Fluor 532 (AF532) and doxorubicin (DOX). The external surface was functionalized with polyethylene glycol (PEG) alone or with the tumor-targeting ligand folic acid (FA) through a PEG linker. The resulting bi-functional nanoparticles remained intact and correctly assembled. The uptake of FA-displaying nanoparticles (D381C-AF532-PEG-FA) by cells overexpressing the folate receptor was approximately six times greater than of non-targeting nanoparticles (D381C-AF532-PEG) and was confirmed to be FA-specific. Nanoparticles containing DOX were all cytotoxic in the low micromolar range. To our knowledge, this work is the first time that acid-labile drug release and folate receptor targeting have been simultaneously integrated onto recombinant protein nanoparticles, and it demonstrates the potential of using biofabrication strategies to generate functional nanomaterials.

Antimitochondrial Antibody Recognition and Structural Integrity of the Inner Lipoyl Domain of the E2 Subunit of Pyruvate Dehydrogenase Complex

Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl β-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a (5)Ile→Ala and (12)Ile→Ala double mutant. Single alanine replacement at (5)Ile, (12)Ile, and (15)Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at (5)Ile, (12)Ile, and (15)Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the (5)Ile and (12)Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl β-sheet structural conformation in anti-PDC-E2 recognition.

Antimitochondrial antibody heterogeneity and the xenobiotic etiology of primary biliary cirrhosis

Antimitochondrial antibodies (AMAs) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with primary biliary cirrhosis (PBC) and are present before the onset of clinical disease. The recent demonstration that AMAs recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2 raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly immunoglobulin M (IgM) and using sera from a large cohort of PBC patients and controls (n = 516), we examined in detail sera reactivity against either 6,8-bis(acetylthio) octanoic acid (SAc)-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition enzyme-linked immunosorbent assay (ELISA); SAc conjugate and rPDC-E2-specific affinity-purified antibodies were also examined for antigen specificity, isotype, and crossreactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, crossreactivity, and inhibition studies. In both early and late-stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc than to rPDC-E2 in early-stage versus late-stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. Specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e., by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Biomimetic Design of Protein Nanomaterials for Hydrophobic Molecular Transport

Biomaterials such as self-assembling biological complexes have demonstrated a variety of applications in materials science and nanotechnology. The functionality of protein-based materials, however, is often limited by the absence or locations of specific chemical conjugation sites. In this investigation, we developed a new strategy for loading organic molecules into the hollow cavity of a protein nanoparticle that relies only on non-covalent interactions, and we demonstrated its applicability in drug delivery. Based on a biomimetic model that incorporates multiple phenylalanines to create a generalized binding site, we retained and delivered the antitumor compound doxorubicin by redesigning a caged protein scaffold. Through an iterative combination of structural modeling and protein engineering, we obtained new variants of the E2 subunit of pyruvate dehydrogenase with varying levels of drug-carrying capabilities. We found that an increasing number of introduced phenylalanines within the scaffold cavity generally resulted in greater drug loading capacities. Drug loading levels could be achieved that were greater than conventional nanoparticle delivery systems. These protein nanoparticles containing doxorubicin were taken up by breast cancer cells and induced significant cell death. Our novel approach demonstrates a universal strategy to design de novo hydrophobic binding domains within protein-based scaffolds for molecular encapsulation and transport, and it broadens the ability to attach guest molecules to this class of materials.

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.

PH-Triggered Disassembly in a Caged Protein Complex

Self-assembling protein cage structures have many potential applications in nanotechnology, one of which is therapeutic delivery. For intracellular targeting, pH-controlled disassembly of virus-like particles and release of their molecular cargo is particularly strategic. We investigated the potential of using histidines for introducing pH-dependent disassembly in the E2 subunit of pyruvate dehydrogenase. Two subunit interfaces likely to disrupt stability, an intratrimer interface (the N-terminus) and an intertrimer interface (methionine-425), were redesigned. Our results show that changing the identity of the putative anchor site 425 to histidine does not decrease stability. In contrast, engineering non-native pH-dependent behavior and modulating the transition pH at which disassembly occurs can be accomplished by mutagenesis of the N-terminus and by ionic strength changes. The observed pH-triggered disassembly is due to electrostatic repulsions generated by histidine protonation. These results suggest that altering the degree of electrostatic repulsion at subunit interfaces could be a generally applicable strategy for designing pH-triggered assembly in protein macromolecular structures.

Lipoic Acid Synthesis and Attachment in Yeast Mitochondria

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of alpha-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.

Design of a pH-Dependent Molecular Switch in a Caged Protein Platform

Self-assembling protein cages provide a wide range of possible applications in nanotechnology. We report the first example of an engineered pH-dependent molecular switch in a virus-like particle. By genetically manipulating the subunit-subunit interface of the E2 subunit of pyruvate dehydrogenase, we introduce pH-responsive assembly into a scaffold that is natively stable at both pH 5.0 and 7.4. The redesigned protein module yields an intact, stable particle at pH 7.4 that dissociates at pH 5.0. This triggered behavior is especially relevant for applications in therapeutic delivery.

Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis.

Anti-M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha-ketoacid dehydrogenase complex of the inner mitochondrial membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC), 70 kD; protein X of the PDC, 56 kD; alpha-ketoglutarate dehydrogenase complex, 52 kD; branched-chain alpha-ketoacid dehydrogenase, 52 kD; E1 alpha subunit of PDC, 45 kD; and E1 beta-subunit of PDC, 36 kD). These epitopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria. The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera. However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC-E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the recombinant PDC-E2. In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC-specific epitope at 70 kD. Naturally occurring mitochondrial antibodies (NOMA) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders. They recognized either a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta). None of the sera from 100 blood donors had anti-M2 but 14 had NOMA. Testing anti-M2 and NOMA-positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E. coli it could be shown that--like mammalian mitochondria--they contain both the PBC-specific M2 antigen as well as the non-PBC-specific naturally occurring mitochondrial antigen system (NOMAg). The observation that PBC-specific antibodies were preferentially induced in patients suffering from a mycobacterial infection may provide some new clues to the still unknown etiology of PBC.