Human Mitochondrial Enzyme Research Articles

Mt-LAF3 is a pseudouridine synthase ortholog required for mitochondrial rRNA and mRNA gene expression in Trypanosoma brucei

Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. Trypanosoma brucei mitochondrial (mt)-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null (CN) for mt-LAF3 expression and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential (ΔΨm). Addition of a mutant gamma ATP synthase allele to the CN cells permitted ΔΨm maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of ΔΨm and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold.

Pathogenicity Analysis of a Novel Variant in GTPBP3 Causing Mitochondrial Disease and Systematic Literature Review

Defect of GTPBP3, the human mitochondrial tRNA-modifying enzyme, can lead to Combined Oxidative Phosphorylation Deficiency 23 (COXPD23). Up to now, about 20 different variants of the GTPBP3 gene have been reported; however, genotype-phenotype analysis has rarely been described. Here, we reported a 9-year-old boy with COXPD23 who presented with hyperlactatemia, hypertrophic cardiomyopathy, seizures, feeding difficulties, intellectual disability and motor developmental delay, and abnormal visual development. Biallelic pathogenic variants of the GTPBP3 gene were identified in this boy, one novel variant c.1102dupC (p. Arg368Profs*22) inherited from the mother and the other known variant c.689A>C (p. Gln230Pro) inherited from father. We curated 18 COXPD23 patients with GTPBP3 variants to investigate the genotype-phenotype correlation. We found that hyperlactatemia and cardiomyopathy were critical clinical features in COXPD23 and the average onset age was 1.7 years (3 months of age for the homozygote). Clinical classification of COXPD23 for the two types, severe and mild, was well described in this study. We observed arrhythmia and congestive heart failure frequently in the severe type with early childhood mortality, while developmental delay was mainly observed in the mild type. The proportion of homozygous variants (71.4%) significantly differed from that of compound heterozygous variants (18.1%) in the severe type. Compared with the variants in gnomAD, the proportion of LOFVs in GTPBP3 was higher in COXPD23 patients (48.6% versus 8.9%, p < 0.0001 ****), and 31% of them were frameshift variants, showing the LOF mechanism of GTPBP3. Additionally, the variants in patients were significantly enriched in the TrmE-type G domain, indicating that the G domain was crucial for GTPBP3 protein function. The TrmE-type G domain contained several significant motifs involved in the binding of guanine nucleotides and Mg2+, the hydrolysis of GTP, and the regulation of the functional status of GTPases. In conclusion, we reported a mild COXPD23 case with typical GTPBP3-related symptoms, including seizures and abnormal visual development seldom observed previously. Our study provides novel insight into understanding the clinical diagnosis and genetic counseling of patients with COXPD23 by exploring the genetic pathogenesis and genotype-phenotype correlation of COXPD23.

The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids

The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzyme's catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9',C10'. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Safety, Tolerability and Pharmacokinetics of Vidofludimus calcium (IMU-838) After Single and Multiple Ascending Oral Doses in Healthy Male Subjects

Background and ObjectiveVidofludimus is a potent and selective inhibitor of human mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The clinical efficacy and safety profile of vidofludimus has been analyzed in patients suffering from rheumatoid arthritis and Crohn’s disease and ulcerative colitis. In previous sudies, hematuria at higher doses occurred in close temporal relationship to vidofludimus administration and appeared to be dose related. The present report describes the results from two phase 1 studies conducted in healthy male subjects to investigate the safety, tolerability and pharmacokinetics after single and multiple ascending (SAD and MAD) oral doses of IMU-838 (vidofludimus calcium, tablets containing a specific polymorph). The effect of food on the pharmacokinetics of IMU-838 was also assessed in the SAD study.MethodsIn the SAD study, 12 subjects received single doses of IMU-838 under fasting (10–40 mg) or fed (10 mg) condition in an open-label, partial parallel group design. In the MAD study, 52 subjects received multiple doses of IMU-838 (30–50 mg) in a double-blind, placebo-controlled, parallel group design.ResultsIMU-838 showed dose-proportional pharmacokinetics after single and multiple oral dosing in both SAD and MAD studies. IMU-838 was well absorbed after single daily doses. Food did not impact the pharmacokinetics of IMU-838. The accumulation factor for multiple daily dosing was approximately 2. Steady-state concentrations were reached within about 6–8 days for 30–50 mg groups. The geometric mean plasma half-life of IMU-838 at steady state was approximately 30 h, which supports its use for once-daily dosing regimen. Single and multiple oral doses of IMU-838 were safe and well tolerated.ConclusionOverall, oral IMU-838 was generally well tolerated in SAD and MAD studies in healthy subjects over a wide dose range of 10–50 mg. IMU-838 was well absorbed after single daily doses. IMU-838 showed dose proportional pharmacokinetics after single and multiple oral dosing.Electronic supplementary materialThe online version of this article (10.1007/s13318-020-00623-7) contains supplementary material, which is available to authorized users.

Pathogenic Mutations in a Human Mitochondrial Enzyme Affect Protein Stability

Mutations in human mitochondrial alanyl tRNA‐synthetase have been correlated with at least two different pathologies, infantile cardiomyopathy and leukodystrophy. We have used a bacterial expression system to study the effects of these mutations on protein stability. All mutations tested lead to reduced amounts of full‐length soluble enzyme. Several render the protein insoluble, while others lead to proteolysis into soluble or insoluble fragments. Correlations between protein stability and disease state will be presented. We are currently investigating whether the addition of solubility tags will reverse these effects and facilitate the purification of full‐length mutants.Support or Funding InformationCarleton College and the Towsley FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase - extensive moonlighting in mitochondrial tRNA biogenesis.

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5 0 extensions, is the methyltransferase responsible for m 1 G9 and m 1 A9 formation. The ability of the mitochondrial tRNA:m 1 R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5 0 end processing.

Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2.

Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.

The crystal structure of the drug targetMycobacterium tuberculosismethionyl-tRNA synthetase in complex with a catalytic intermediate

Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.

Eye diseases identified in the ENU‐Ageing Screen

SummaryMRC Harwell is generating cohorts of mutagenized mice, bred to reveal recessive mutations. Cohorts are repeatedly screened for a range of phenotypes at different ages up to 18 months. Included among the screens is optokinetic response to test visual acuity, pupillometry and examination by slit lamp and indirect ophthalmoscopy. We identify both early and late onset disease models, and document disease progression over the lifetime of the mouse. The genetic design of the screen means that the mutant gene can be rapidly identified by mapping and next‐generation sequencing To date we have identified early onset phenotypes including retinal degeneration due to mutation of Rpgrip1, a model for Leber congenital amaurosis‐6, and abnormal pupil response due to mutation in Chrm3. Late onset phenotypes include corneal opacity, caused by mutation of Ikzf2, and progressive vision loss with retinal degeneration. We have identified a missense mutation in Idh3a that results in reduced visual acuity and retinal degeneration from 12 months. Idh3a encodes the α subunit of the mitochondrial enzyme, isocitrate dehydrogenase. This mouse is a model for retinitis pigmentosa 46, due to mutation in the β subunit of human mitochondrial IDH enzyme.

CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown.

P74: Novel human mitochondrial enzyme can transform nitrite into nitric oxide

Endogenous metabolism of nitrate to nitrite to nitric oxide (NO) affects numerous biological events in humans, such as blood pressure control and hypoxic vasodilation, yet the molecular mechanism of this transformation remains uncertain and controversial. Nitrate and nitrite conversion to NO are reductive processes, requiring electron and proton transfer reactions, suggesting that oxidoreductase enzymes are involved. Nitrate reduction may occur through a combination of endogenous (i.e. human enzymes) and exogenous (i.e. commensal bacteria) nitrate reductase enzymes, while nitrite reduction to nitric oxide is likely catalyzed by endogenous nitrite reductase enzymes. Several mechanisms have been investigated, yet no conclusive data are available to support a single enzyme as an indispensable component for reduction of nitrite to NO in human tissue. It is likely that different enzyme systems are active in specific tissues under differential hypoxia, pH and co-factor regulatory control. We have identified a novel human nitrite reductase enzyme, which we hypothesize may contribute to mitochondrial reduction of nitrite to NO. The mitochondria amidoxime reducing component (mARC) enzyme is widely expressed in human tissue, but has an undefined physiological function. We hypothesize that mARC catalyzes the NADH-dependent reduction of nitrite to NO. To test this hypothesis the kinetics of nitrite reduction to NO by human mARC was investigated. Recombinant mARC was generated using standard molecular biology techniques, and then isolated using metal affinity chromatography. Site directed mutagenesis was used to change the active site cysteine into alanine. Human cytochrome b5 and cytochrome b5 reductase were also isolated to determine if mARC can utilize NADH as an electron source in conjunction with these enzymes. Nitric oxide chemiluminescence spectroscopy was used to measure NO-formation rates under anaerobic conditions. Our study established that mARC can generate NO from nitrite in the presence of NADH, cytochrome b5, and cytochrome b5 reductase at pH 7.4. The maximum velocity (Vmax) of NO-formation measured was 5 nmoles NO s−1 mg−1 protein. Moreover, mutation of the putative active site cysteine residue to alanine, and substitution of tungsten for molybdenum, completely abolished enzyme activity. The kinetic data supports our hypothesis and establishes that human mARC is capable of catalyzing reduction of nitrite to NO. Moreover, these data suggest that cysteine 270 and molybdenum are important in the transformation of nitrite to NO. Disclosure Supported by institution training grant (T32).

Enteroviruses and the pathogenesis of type 1 diabetes revisited: cross‐reactivity of enterovirus capsid protein (VP1) antibodies with human mitochondrial proteins

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m1G9 and m1A9 formation. The ability of the mitochondrial tRNA:m1R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.

Nuclear RNase P of Trypanosoma brucei: A Single Protein in Place of the Multicomponent RNA-Protein Complex

SummaryRNase P is the endonuclease that removes 5′ extensions from tRNA precursors. In its best-known form, the enzyme is composed of a catalytic RNA and a protein moiety variable in number and mass. This ribonucleoprotein enzyme is widely considered ubiquitous and apparently reached its highest complexity in the eukaryal nucleus, where it is typically composed of at least ten subunits. Here, we show that in the protist Trypanosoma brucei, two proteins are the sole forms of RNase P. They localize to the nucleus and the mitochondrion, respectively, and have RNase P activity each on their own. The protein-RNase P is, moreover, capable of replacing nuclear RNase P in yeast cells. This shows that complex ribonucleoprotein structures and RNA catalysis are not necessarily required to support tRNA 5′ end formation in eukaryal cells.

Exploiting Protein Engineering and Crystal Polymorphism for Successful X-ray Structure Determination

The preparation of high quality crystals is a central issue in the process of determining 3D structures of biomolecules by X-ray crystallography. The success of this key step frequently depends upon the right choice and the rigorous characterization of the target. Further, the identification and refinement of the growth conditions of a supplementary crystalline polymorph may be profitable. Four representative examples illustrate how the critical parameters can be handled. These case studies include chemically and structurally different biological entities: bacterial RNA chaperone Hfq, human mitochondrial enzyme tyrosyl-tRNA synthetase, yeast exosome subcomplex, and icosahedral virus causing grapevine fanleaf disease. The winding paths which led to the determination of each structure at atomic resolution are described together with related crystallogenesis strategies.

523: Metabolism of 17-hydroxyprogesteronecaproate (17-P) by human placental mitochondrial and microsomal enzymes

523: Metabolism of 17-hydroxyprogesteronecaproate (17-P) by human placental mitochondrial and microsomal enzymes

Human liver mitochondrial cytochrome P450 2D6 – individual variations and implications in drug metabolism

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.

Toward the Full Set of Human Mitochondrial Aminoacyl-tRNA Synthetases: Characterization of AspRS and TyrRS

The human mitochondrion possesses a translational machinery devoted to the synthesis of 13 proteins. While the required tRNAs and rRNAs are produced by transcription of the mitochondrial genome, all other factors needed for protein synthesis are synthesized in the cytosol and imported. This is the case for aminoacyl-tRNA synthetases, the enzymes which esterify their cognate tRNA with the specific amino acid. The genes for the full set of cytosolic aaRSs are well defined, but only nine genes for mitochondrial synthetases are known. Here we describe the genes for human mitochondrial aspartyl- and tyrosyl-tRNA synthetases and the initial characterization of the enzymes. Both belong to the expected class of synthetases, have a dimeric organization, and aminoacylate Escherichia coli tRNAs as well as in vitro transcribed human mitochondrial tRNAs. Genes for the remaining missing synthetases were also found with the exception of glutaminyl-tRNA synthetase. Their sequence analysis confirms and further extends the view that, except for lysyl- and glycyl-tRNA synthetases, human mitochondrial and cytosolic enzymes are coded by two different sets of genes.

Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics.

Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics.

Dual functional roles of ATP in the human mitochondrial malic enzyme.

Human mitochondrial malic enzyme is a regulatory enzyme with ATP as an inhibitor. Structural studies reveal that the enzyme has two ATP-binding sites, one at the NAD(+)-binding site in the active center and the other at the exo site in the tetramer interface. Inhibition of the enzyme activity is due to the competition between ATP and NAD(+) for the nucleotide-binding site at the active center with an inhibition constant of 81 microM. Binding of the ATP molecule at the exo site, on the other hand, is important for the maintenance of the quaternary structural integrity. The enzyme exists in solution at neutral pH and at equilibrium of the dimer and tetramer with a dissociation constant (K(TD)) of 0.67 microM. ATP, at a physiological concentration, shifts the equilibrium toward tetramer and decreases the K(TD) by many orders of magnitude. Mutation of a single residue Arg542 at the tetrameric interfacial exo site resulted in dimeric mutants. ATP thus has dual functional roles in the mitochondrial malic enzyme.