Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

Background: Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail. Results: We demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects. Conclusion: Together, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis

Thermosensory perception regulates speed of movement in response to temperature changes in <i>Drosophila melanogaster</i>

Temperature influences physiology and behavior of all organisms. For ectotherms, which lack central temperature regulation, temperature adaptation requires sheltering from or moving to a heat source. As temperature constrains the rate of metabolic reactions, it can directly affect ectotherm physiology and thus behavioral performance. This direct effect is particularly relevant for insects whose small body readily equilibrates with ambient temperature. In fact, models of enzyme kinetics applied to insect behavior predict performance at different temperatures, suggesting that thermal physiology governs behavior. However, insects also possess thermosensory neurons critical for locating preferred temperatures, showing cognitive control. This suggests that temperature-related behavior can emerge directly from a physiological effect, indirectly as consequence of thermosensory processing, or through both. To separate the roles of thermal physiology and cognitive control, we developed an arena that allows fast temperature changes in time and space, and in which animals' movements are automatically quantified. We exposed wild-type and thermosensory receptor mutants Drosophila melanogaster to a dynamic temperature environment and tracked their movements. The locomotor speed of wild-type flies closely matched models of enzyme kinetics, but the behavior of thermosensory mutants did not. Mutations in thermosensory receptor dTrpA1 (Transient receptor potential) expressed in the brain resulted in a complete lack of response to temperature changes, while mutation in peripheral thermosensory receptor Gr28b(D) resulted in diminished response. We conclude that flies react to temperature through cognitive control, informed by interactions between various thermosensory neurons, whose behavioral output resembles that of enzyme kinetics

Coenzyme A levels influence protein acetylation, CoAlation and 4'-phosphopantetheinylation:Expanding the impact of a metabolic nexus molecule

Coenzyme A (CoA) is a key molecule in cellular metabolism including the tricarboxylic acid cycle, fatty acid synthesis, amino acid synthesis and lipid metabolism. Moreover, CoA is required for biological processes like protein post-translational modifications (PTMs) including acylation. CoA levels affect the amount of histone acetylation and thereby modulate gene expression. A direct influence of CoA levels on other PTMs, like CoAlation and 4'-phosphopantetheinylation has been relatively less addressed and will be discussed here. Increased CoA levels are associated with increased CoAlation, whereas decreased 4'-phosphopantetheinylation is observed under circumstances of decreased CoA levels. We discuss how these two PTMs can positively or negatively influence target proteins depending on CoA levels. This review highlights the impact of CoA levels on post-translational modifications, their counteractive interplay and the far-reaching consequences thereof

De novo formation of centrosomes in vertebrate cells arrested during S phase

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting “maternal” centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: ∼5–8 h after ablation, clouds of pericentriolar material (PCM) containing γ-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2–14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions

Specific protein homeostatic functions of small heat-shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat-shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress-denatured substrates and/or to prevent aggregation of disease-associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70-dependent refolding of stress-denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70-independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo

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

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

Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2(−/−)) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2(−/−) mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration

Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G2/M checkpoint control in Drosophila cells

North Sea Progressive Myoclonus Epilepsy is Exacerbated by Heat, A Phenotype Primarily Associated with Affected Glia

Progressive myoclonic epilepsies (PMEs) comprise a group of rare disorders of different genetic aetiologies, leading to childhood-onset myoclonus, myoclonic seizures and subsequent neurological decline. One of the genetic causes for PME, a mutation in the gene coding for Golgi SNAP receptor 2 (GOSR2), gives rise to a PME-subtype prevalent in Northern Europe and hence referred to as North Sea Progressive Myoclonic Epilepsy (NS-PME). Treatment for NS-PME, as for all PME subtypes, is symptomatic; the pathophysiology of NS-PME is currently unknown, precluding targeted therapy. Here, we investigated the pathophysiology of NS-PME. By means of chart review in combination with interviews with patients (n = 14), we found heat to be an exacerbating factor for a majority of NS-PME patients (86%). To substantiate these findings, we designed a NS-PME Drosophila melanogaster model. Downregulation of the Drosophila GOSR2-orthologue Membrin leads to heat-induced seizure-like behaviour. Specific downregulation of GOSR2/Membrin in glia but not in neuronal cells resulted in a similar phenotype, which was progressive as the flies aged and was partially responsive to treatment with sodium barbital. Our data suggest a role for GOSR2 in glia in the pathophysiology of NS-PME