RESUMO
Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.
Assuntos
Coenzima A , Microbiota , Animais , Coenzima A/genética , Coenzima A/metabolismo , Drosophila/metabolismo , Feminino , Humanos , Mães , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Zigoto/metabolismoRESUMO
Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.
RESUMO
Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.
Assuntos
Apoptose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Núcleo Celular/metabolismo , Regulação para Baixo , Drosophila melanogaster/ultraestrutura , Retículo Endoplasmático/metabolismo , Feminino , Fertilidade , Mutação/genética , Oogênese , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , FenótipoRESUMO
Adult stem cells are often touted as therapeutic agents in the regenerative medicine field, however data detailing both the engraftment and functional capabilities of solid tissue derived human adult epithelial stem cells is scarce. Here we show the isolation of adult human salivary gland (SG) stem/progenitor cells and demonstrate at the single cell level in vitro self-renewal and differentiation into multilineage organoids. We also show in vivo functionality, long-term engraftment, and functional restoration in a xenotransplantation model. Indeed, transplanted human salisphere-derived cells restored saliva production and greatly improved the regenerative potential of irradiated SGs. Further selection for c-Kit expression enriched for cells with enhanced regenerative potencies. Interestingly, interaction of transplanted cells with the recipient SG may also be involved in functional recovery. Thus, we show for the first time that salispheres cultured from human SGs contain stem/progenitor cells capable of self-renewal and differentiation and rescue of saliva production. Our study underpins the therapeutic promise of salisphere cell therapy for the treatment of xerostomia.
Assuntos
Proteínas Proto-Oncogênicas c-kit/biossíntese , Glândulas Salivares/citologia , Transplante de Células-Tronco , Xerostomia/terapia , Animais , Diferenciação Celular/genética , Diferenciação Celular/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-kit/genética , Radiação , Glândulas Salivares/metabolismo , Glândulas Salivares/transplante , Análise de Célula Única , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação , Xerostomia/patologiaRESUMO
The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.
Assuntos
Caenorhabditis elegans/metabolismo , Coenzima A/biossíntese , Drosophila/metabolismo , Panteteína/análogos & derivados , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Linhagem Celular , Coenzima A/sangue , Coenzima A/farmacologia , Coenzima A Ligases/metabolismo , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Feminino , Células HEK293 , Humanos , Longevidade/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Panteteína/sangue , Panteteína/metabolismo , Panteteína/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease modelsâespecially 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 pathwayâphosphopantothenoylcysteine synthetase (PPCS)âthrough 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 enzymesâincluding the elusive binding mode for cysteineâand 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.
Assuntos
Coenzima A/metabolismo , Inibidores Enzimáticos/farmacologia , Ácido Pantotênico/análogos & derivados , Peptídeo Sintases/antagonistas & inibidores , Ácido gama-Aminobutírico/análogos & derivados , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Cristalização , Drosophila melanogaster , Cinética , Conformação Molecular , Ácido Pantotênico/farmacologia , Peptídeo Sintases/metabolismo , Especificidade por Substrato , Ácido gama-Aminobutírico/farmacologiaRESUMO
PKAN, CoPAN, MePAN, and PDH-E2 deficiency share key phenotypic features but harbor defects in distinct metabolic processes. Selective damage to the globus pallidus occurs in these genetic neurodegenerative diseases, which arise from defects in CoA biosynthesis (PKAN, CoPAN), protein lipoylation (MePAN), and pyruvate dehydrogenase activity (PDH-E2 deficiency). Overlap of their clinical features suggests a common molecular etiology, the identification of which is required to understand their pathophysiology and design treatment strategies. We provide evidence that CoA-dependent activation of mitochondrial acyl carrier protein (mtACP) is a possible process linking these diseases through its effect on PDH activity. CoA is the source for the 4'-phosphopantetheine moiety required for the posttranslational 4'-phosphopantetheinylation needed to activate specific proteins. We show that impaired CoA homeostasis leads to decreased 4'-phosphopantetheinylation of mtACP. This results in a decrease of the active form of mtACP, and in turn a decrease in lipoylation with reduced activity of lipoylated proteins, including PDH. Defects in the steps of a linked CoA-mtACP-PDH pathway cause similar phenotypic abnormalities. By chemically and genetically re-activating PDH, these phenotypes can be rescued, suggesting possible treatment strategies for these diseases.
Assuntos
Proteína de Transporte de Acila/metabolismo , Coenzima A/metabolismo , Doenças Neurodegenerativas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína de Transporte de Acila/genética , Animais , Western Blotting , Linhagem Celular , Drosophila , Feminino , Citometria de Fluxo , Células HEK293 , Humanos , Masculino , Doenças Neurodegenerativas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
The VPS13A gene is associated with the neurodegenerative disorder Chorea Acanthocytosis. It is unknown what the consequences are of impaired function of VPS13A at the subcellular level. We demonstrate that VPS13A is a peripheral membrane protein, associated with mitochondria, the endoplasmic reticulum and lipid droplets. VPS13A is localized at sites where the endoplasmic reticulum and mitochondria are in close contact. VPS13A interacts with the ER residing protein VAP-A via its FFAT domain. Interaction with mitochondria is mediated via its C-terminal domain. In VPS13A-depleted cells, ER-mitochondria contact sites are decreased, mitochondria are fragmented and mitophagy is decreased. VPS13A also localizes to lipid droplets and affects lipid droplet motility. In VPS13A-depleted mammalian cells lipid droplet numbers are increased. Our data, together with recently published data from others, indicate that VPS13A is required for establishing membrane contact sites between various organelles to enable lipid transfer required for mitochondria and lipid droplet related processes.
Assuntos
Retículo Endoplasmático/genética , Gotículas Lipídicas/metabolismo , Mitocôndrias/genética , Proteínas de Transporte Vesicular/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Neuroacantocitose/genética , Doenças Neurodegenerativas/genética , Domínios Proteicos , Proteínas de Transporte Vesicular/metabolismoRESUMO
Pantothenate kinase-associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease-vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4'-phosphopantetheine, normalized levels of the CoA-, iron-, and dopamine-related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4'-phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron-sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4'-phosphopantetheine as a candidate therapeutic for PKAN.
Assuntos
Coenzima A/metabolismo , Dopamina/metabolismo , Ferro/metabolismo , Panteteína/análogos & derivados , Neurodegeneração Associada a Pantotenato-Quinase/tratamento farmacológico , Neurodegeneração Associada a Pantotenato-Quinase/metabolismo , Animais , Biomarcadores/metabolismo , Genótipo , Camundongos , Panteteína/farmacologia , Panteteína/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis.
Assuntos
Encéfalo/fisiologia , Proteínas de Drosophila/fisiologia , Homeostase/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Encéfalo/patologia , Drosophila , Proteínas de Drosophila/genética , Humanos , Mutação , Proteínas de Transporte Vesicular/genéticaRESUMO
Coenzyme A is an essential metabolite known for its central role in over one hundred cellular metabolic reactions. In cells, Coenzyme A is synthesized de novo in five enzymatic steps with vitamin B5 as the starting metabolite, phosphorylated by pantothenate kinase. Mutations in the pantothenate kinase 2 gene cause a severe form of neurodegeneration for which no treatment is available. One therapeutic strategy is to generate Coenzyme A precursors downstream of the defective step in the pathway. Here we describe the synthesis, characteristics and in vivo rescue potential of the acetyl-Coenzyme A precursor S-acetyl-4'-phosphopantetheine as a possible treatment for neurodegeneration associated with pantothenate kinase deficiency.
Assuntos
Transtornos Heredodegenerativos do Sistema Nervoso/tratamento farmacológico , Panteteína/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Soro/química , Animais , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Humanos , Camundongos , Panteteína/administração & dosagem , Panteteína/síntese química , Panteteína/isolamento & purificação , Panteteína/farmacocinética , Resultado do TratamentoRESUMO
INTRODUCTION: During radiotherapy salivary glands of head and neck cancer patients are unavoidably co-irradiated, potentially resulting in life-long impairment. Recently we showed that transplantation of salisphere-derived c-Kit expressing cells can functionally regenerate irradiated salivary glands. This study aims to select a more potent subpopulation of c-Kit(+) cells, co-expressing stem cell markers and to investigate whether long-term tissue homeostasis is restored after stem cell transplantation. METHODS AND RESULTS: Salisphere derived c-Kit(+) cells that co-expressed CD24 and/or CD49f markers, were intra-glandularly injected into 15 Gy irradiated submandibular glands of mice. Particularly, c-Kit(+)/CD24(+)/CD49f(+) cell transplanted mice improved saliva production (54.59 ± 11.1%) versus the irradiated control group (21.5 ± 8.7%). Increase in expression of cells with differentiated duct cell markers like, cytokeratins (CK8, 18, 7 and 14) indicated functional recovery of this compartment. Moreover, ductal stem cell marker expression like c-Kit, CD133, CD24 and CD49f reappeared after transplantation indicating long-term functional maintenance potential of the gland. Furthermore, a normalization of vascularization as indicated by CD31 expression and reduction of fibrosis was observed, indicative of normalization of the microenvironment. CONCLUSIONS: Our results show that stem cell transplantation not only rescues hypo-salivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue.
Assuntos
Proteínas Proto-Oncogênicas c-kit/fisiologia , Lesões por Radiação/terapia , Glândulas Salivares/efeitos da radiação , Transplante de Células-Tronco , Animais , Antígeno CD24/análise , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Homeostase , Integrina alfa6/análise , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/análise , Regeneração , Glândulas Salivares/fisiologiaRESUMO
Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process. In the manifestation of diseases such as Sjögren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer. The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.
Assuntos
Técnicas Citológicas/métodos , Glândulas Salivares/citologia , Células-Tronco/citologia , Animais , Humanos , CamundongosRESUMO
BACKGROUND: Stem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patient's quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland. METHODS: Mouse submandibular gland tissue, isolated cells and cultured 3 day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15 Gy and transplanted with cells expressing defined markers. RESULTS: Cells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function. CONCLUSIONS: Different stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.
Assuntos
Glândulas Salivares/citologia , Glândulas Salivares/efeitos da radiação , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Células-Tronco/efeitos da radiação , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Antígeno CD24/metabolismo , Feminino , Citometria de Fluxo , Glicoproteínas/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Técnicas Imunoenzimáticas , Integrina alfa6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Regeneração , Células-Tronco/metabolismo , Xerostomia/etiologia , Xerostomia/terapiaRESUMO
BACKGROUND: Recently, we showed that transplantation of 100-300 c-Kit(+) stem cells isolated from cultured salispheres ameliorates radiation-damage in murine salivary glands. The aim of this study is to optimize and translate these findings from mice to man. METHODS: Mouse and human non-malignant parotid and submandibular salivary gland tissue was collected and enzymatically digested. The remaining cell suspension was cultured according to our salisphere culture method optimized for murine salispheres. Salisphere cells were tested using 3D matrix culturing for their in vitro stem cell characteristics such as the potential to differentiate into tissue specific cell types. Several potential mouse and human salivary gland stem cells were selected using FACS. RESULTS: In human salivary gland, c-Kit(+) cells were only detected in excretory ducts as shown previously in mice. From both human parotid and submandibular gland cell suspensions salispheres could be grown, which when placed in 3D culture developed ductal structures and mucin-expressing acinar-like cells. Moreover, cells dispersed from primary salispheres were able to form secondary spheres in matrigel, a procedure that could be repeated for at least seven passages. Approximately 3000 c-Kit+ cells could be isolated from primary human salispheres per biopsy. CONCLUSION: Human salivary glands contain a similar 'putative' stem cell population as rodents, expressing c-kit and capable of in vitro differentiation and self-renewal. In the future, these cells may have the potential to reduce radiotherapy-induced salivary gland dysfunction in patients.