393 research outputs found

    Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds

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    Mutations in the pantothenate kinase 2 gene (PANK2) are the cause of pantothenate kinase-associated neurodegeneration (PKAN), the most common form of neurodegeneration with brain iron accumulation. Although different disease models have been created to investigate the pathogenic mechanism of PKAN, the cascade of molecular events resulting from CoA synthesis impairment is not completely understood. Moreover, for PKAN disease, only symptomatic treatments are available. Despite the lack of a neural system, Saccharomyces cerevisiae has been successfully used to decipher molecular mechanisms of many human disorders including neurodegenerative diseases as well as iron-related disorders. To gain insights into the molecular basis of PKAN, a yeast model of this disease was developed: a yeast strain with the unique gene encoding pantothenate kinase CAB1 deleted, and expressing a pathological variant of this enzyme. A detailed functional characterization demonstrated that this model recapitulates the main phenotypes associated with human disease: mitochondrial dysfunction, altered lipid metabolism, iron overload, and oxidative damage suggesting that the yeast model could represent a tool to provide information on pathophysiology of PKAN. Taking advantage of the impaired oxidative growth of this mutant strain, a screening for molecules able to rescue this phenotype was performed. Two molecules in particular were able to restore the multiple defects associated with PKAN deficiency and the rescue was not allele-specific. Furthermore, the construction and characterization of a set of mutant alleles, allowing a quick evaluation of the biochemical consequences of pantothenate kinase (PANK) protein variants could be a tool to predict genotype/phenotype correlation

    iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

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    Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent

    The Coenzyme A Level Modulator Hopantenate (HoPan) Inhibits Phosphopantotenoylcysteine Synthetase Activity

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    The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease models-especially for pantothenate kinase associated neurodegeneration (PKAN), a genetic disease rooted in impaired CoA metabolism. This use of HoPan was based on reports that it inhibits pantothenate kinase (PanK), the first enzyme of CoA biosynthesis. Using a combination of in vitro enzyme kinetic studies, crystal structure analysis, and experiments in a typical PKAN cell biology model, we demonstrate that instead of inhibiting PanK, HoPan relies on it for metabolic activation. Once phosphorylated, HoPan inhibits the next enzyme in the CoA pathway-phosphopantothenoylcysteine synthetase (PPCS)-through formation of a nonproductive substrate complex. Moreover, the obtained structure of the human PPCS in complex with the inhibitor and activating nucleotide analogue provides new insights into the catalytic mechanism of PPCS enzymes-including the elusive binding mode for cysteine-and reveals the functional implications of mutations in the human PPCS that have been linked to severe dilated cardiomyopathy. Taken together, this study demonstrates that the molecular mechanism of action of HoPan is more complex than previously thought, suggesting that the results of studies in which it is used as a tool compound must be interpreted with care. Moreover, our findings provide a clear framework for evaluating the various factors that contribute to the potency of CoA-directed inhibitors, one that will prove useful in the future rational development of potential therapies of both human genetic and infectious diseases

    Pantothenate Kinase 1 Is Required to Support the Metabolic Transition from the Fed to the Fasted State

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    Coenzyme A (CoA) biosynthesis is regulated by the pantothenate kinases (PanK), of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1−/− knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1−/− and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1−/− mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1−/− mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia

    Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target

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    The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells

    Inhibiting the Actions of Essential Biomolecule Phosphopantetheine.

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    Phosphopantetheine is an essential biomolecule required for life. The enzyme phosphopantothenoylcysteine synthetase (PPCS) incorporates the reactive thiol moiety in the Coenzyme A (CoA) biosynthetic pathway. Two types of PPCS exist in bacteria: bifunctional fusion protein Type 1 and monofunctional Type 3. Previously developed cytidylate mimics were determined to have stronger binding affinity for Type 3 Streptococcus pneumoniae PPCS compared to Type 1. To explore the structure activity relationship of Type 3 PPCS, completely conserved residues of bacterial PPCS were probed via saturation mutagenesis in a developed knockout system. Mutagenesis revealed that conserved S. pneumoniae PPCS residues K123 and D93 were stringent, as only K123N/R/M and D93E/N were viable mutations. D93 was determined to be responsible for the association of divalent cations and CTP to the active site, as D93E and D93N exhibited a 1.5-2.7 fold (151-267 μM apparent Km) less affinity for CTP and D93N resulted in a 2-fold drop in MgCl2 affinity. K123 was identified as catalytically significant to the first half CTP utilizing reaction as the K123M and K123R mutations had 1.4-8.8 fold (142-872 μM apparent Km) loss in the binding affinity for CTP. Additionally, phosphopantetheine is the essential prosthetic group of acyl carrier protein (ACP), utilized by gram negative specific acyltransferases (LpxA and LpxD) to construct lipopolysaccharide (LPS). Peptide RJPXD33 (TNLYMLPKWDIP) was discovered to bind and inhibit both LpxA and LpxD (binding constants of 22 μM and 6.5 μM respectively), offering a probe for the development of a dual targeting small molecule inhibitor. The molecular binding mechanism by which RJPXD33 interacts with LpxD was elucidated via fluorescence polarization based binding assays of truncated and alanine-mutated peptide. RJPXD33 P7 was identified to be important to binding LpxD as mutation of the residue to alanine resulted in a binding constant >50 μM, while similar tests in LpxA had a limited effect on binding (9.4 ± 0.6 μM). Residue K8 appeared inconsequential, as mutation to alanine had little effect on binding to LpxA or LpxD (5.1 μM and 3.1 μM binding constants, respectively), but essential to inhibition. Photo-labile RJPXD33 probes were implemented for mapping LpxD-RJPXD33 protein-peptide interactions.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111574/1/heslipky_1.pd
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