RESUMEN
Bacteria are exposed to stresses during their growth and multiplication in their ecological systems to which they respond in multiple ways as expert survivalists. One such response mechanism is to convert to a viable but not culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost their ability to grow on routine growth medium. A large number of bacteria including many pathogenic species have been reported to be able to enter a VBNC state. VBNC differs from culturable cells in various physiological properties which may result in changes in chemical resistance, adhesion, cellular morphology, metabolism, gene expression, membrane and cell wall composition and/or virulence. The ability of VBNC bacteria to return to the culturable state or resuscitate, when the stressor is removed poses a considerable threat to public health. There have been few publications that overtly describe the ability of oral pathogenic species to enter the VBNC state. However, the presence of VBNCs among oral pathogens such as Porphyromonas gingivalis in human chronic infections may be an important virulence factor and have severe implications for therapy. In this review, we intend to i) define and summarize the significance of the VBNC state in general and ii) discuss the VBNC state of oral bacteria with regard to P. gingivalis. Future studies focused on this phenomenon of intraoral VBNC would provide novel molecular insights on the virulence and persistence of oral pathogens during chronic infections and identify potential novel therapies.
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Objective: Microvascular dysfunction is a feature of periodontal disease. P. gingivalis, one of the most common oral bacteria present in gingival tissue biofilms, has also been identified in the gingival capillaries of patients with chronic periodontitis. We sought to determine the effect of P. gingivalis W83 infection on microvascular endothelium in vivo and in vitro. Methods and Results: Interdental papillae of rats with P. gingivalis-induced alveolar bone loss had a more dilated and denser subepithelial capillary network than uninfected controls. P. gingivalis W83 was detected in the epithelial layers, the subepithelial connective tissue matrix, and subgingival capillaries. P. gingivalis invaded human dermal microvascular endothelial cells (HD-MVECS) and persisted up termination (24 h). Colocalization analysis at 2.5, 6, and 24 h post-inoculation showed that 79-88% of internalized bacteria were in ICAM-1 positive endosomes, and 10-39% were in Rab5, Rab7, or LAMP1 positive compartments, but never in autophagosomes. Antibody-based blockade of ICAM-1 significantly reduced W83 invasion in HD-MVECS. P. gingivalis infected HD-MVECS were unable to form vascular networks in Matrigel. Conclusions: P. gingivalis perturbs microvascular endothelial function and invasion of these cells via ICAM-1 may be important for microbial persistence within tissues.
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Ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality after liver surgery. The role of Sirtuin 1 (SIRT1) in hepatic I/R injury remains elusive. Using human and mouse livers, we investigated the effects of I/R on hepatocellular SIRT1. SIRT1 expression was significantly decreased after I/R. Genetic overexpression or pharmacological activation of SIRT1 markedly suppressed defective autophagy, onset of the mitochondrial permeability transition, and hepatocyte death after I/R, whereas SIRT1-null hepatocytes exhibited increased sensitivity to I/R injury. Biochemical approaches revealed that SIRT1 interacts with mitofusin-2 (MFN2). Furthermore, MFN2, but not MFN1, was deacetylated by SIRT1. Moreover, SIRT1 overexpression substantially increased autophagy in wild-type cells, but not in MFN2-deficient cells. Thus, our results demonstrate that the loss of SIRT1 causes a sequential chain of defective autophagy, mitochondrial dysfunction, and hepatocyte death after I/R.
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GTP Fosfohidrolasas/metabolismo , Hígado/irrigación sanguínea , Mitocondrias Hepáticas/enzimología , Sirtuina 1/fisiología , Animales , Autofagia , Calpaína/metabolismo , GTP Fosfohidrolasas/química , Humanos , Isquemia/enzimología , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dominios y Motivos de Interacción de Proteínas , Daño por Reperfusión/enzimologíaRESUMEN
Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-enhanced green fluorescent protein (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titres at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5'-ends of genes from the original data set in which SV2 and UNC93A were identified. Common CREs at the 5'-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins.
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Aedes/virología , Infecciones por Arbovirus/metabolismo , Arbovirus/fisiología , Interacciones Huésped-Patógeno , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Endosomas/metabolismo , Conducta Alimentaria , Silenciador del Gen , Humanos , Ratones , Regiones Promotoras Genéticas , Proteínas Virales/genética , Liberación del Virus , Replicación ViralRESUMEN
INTRODUCTION: Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery. METHODS: By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC). RESULTS: Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h. DISCUSSION: Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.
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Vasos Coronarios/microbiología , Células Endoteliales/microbiología , Endotelio Vascular/microbiología , Streptococcus mutans/fisiología , Estudios de Casos y Controles , Células Cultivadas , Recuento de Colonia Microbiana , Vasos Coronarios/citología , Endotelio Vascular/citología , Humanos , Microscopía Electrónica de Transmisión , Serotipificación , Infecciones Estreptocócicas/microbiología , Streptococcus mutans/clasificaciónAsunto(s)
Lactancia Materna , Ciclohexanoles/farmacocinética , Depresión Posparto/sangre , Leche Humana/química , Adulto , Ciclohexanoles/administración & dosificación , Depresión Posparto/tratamiento farmacológico , Succinato de Desvenlafaxina , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Clorhidrato de VenlafaxinaRESUMEN
Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro- and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.
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Autofagia , Citoplasma/metabolismo , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Proteínas Relacionadas con la Autofagia , Transporte Biológico , División Celular , Membrana Celular/metabolismo , Proteínas de la Membrana , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Peroxisomas/metabolismo , Pichia/citología , Pichia/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Fenproporex is a sympathomimetic agent with a pharmacological profile similar to that of amphetamine. It is available in many countries throughout the world, but it is currently not available in the United States. Because of its stimulant effects, it has a great potential for abuse. To the best of our knowledge, there have been no literature reports of blood or serum concentrations found in therapeutic, toxic, or fatal cases. We report a case where fenproporex was a finding in the death of a young adult. Blood, urine, and gastric contents were analyzed. The following drug concentrations were found: 0.90 mg/L (inferior vena cava blood), 1.2 mg/L (urine), and 120 mg total (gastric) for fenproporex and 0.084 mg/L (inferior vena cava blood), 0.94 mg/L (urine), and 0.14 mg total (gastric) for amphetamine. In addition to the fenproporex, other medications detected and their blood concentrations found in this case were H diazepam (0.54 mg/L), nordiazepam (0.46 mg/L), diphenhydramine (0.12 mg/L), and gamma hydroxybutyric acid (GHB) (1100 mg/L).
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Anfetaminas/envenenamiento , Depresores del Apetito/envenenamiento , Adulto , Anfetaminas/farmacocinética , Antialérgicos/sangre , Anticonvulsivantes/sangre , Depresores del Apetito/farmacocinética , Autopsia , Diazepam/sangre , Difenhidramina/sangre , Sobredosis de Droga , Femenino , Humanos , Hidroxibutiratos/sangre , Nordazepam/sangre , Estómago/química , SuicidioRESUMEN
Cells are capable of adapting to changes in their environment by synthesizing needed proteins and degrading superfluous ones. Pichia pastoris synthesizes peroxisomal enzymes to grow in methanol medium. Upon adapting from methanol medium to one containing glucose, this yeast rapidly and selectively degrades peroxisomes by an autophagic process referred to as pexophagy. In this study, we have utilized a novel approach to identify genes required for this degradative pathway. Our approach involves the random integration of a vector containing the Zeocin resistance gene into the yeast genome by restriction enzyme-mediated integration. Cells unable to degrade peroxisomes during glucose adaptation were isolated, and the genes that were disrupted by the insertion of the vector were determined by sequencing. By using this approach, we have identified a number of genes required for glucose-induced selective autophagy of peroxisomes (GSA genes). We report here the characterization of Gsa11, a unique 208-kDa protein. We found that this protein is required for glucose-induced pexophagy and starvation-induced autophagy. Gsa11 is a cytosolic protein that becomes associated with one or more structures situated near the vacuole during glucose adaptation. The punctate localization of Gsa11 was not observed in gsa10, gsa12, gsa14, and gsa19 mutants. We have previously shown that Gsa9 appears to relocate from a compartment at the vacuole surface to regions between the vacuole and the peroxisomes being sequestered. In the gsa11 mutants, the vacuole only partially surrounded the peroxisomes, but Gsa9 was still distributed around the peroxisome cluster. This suggests that Gsa9 binds to the peroxisomes independent of the vacuole. The data also indicate that Gsa11 is not necessary for Gsa9 to interact with peroxisomes but acts at an intermediate event required for the vacuole to engulf the peroxisomes.
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Proteínas Fúngicas/fisiología , Glucosa/farmacología , Pichia/química , Secuencia de Aminoácidos , Autofagia , Citoplasma/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Peroxisomas/metabolismo , Vacuolas/metabolismoRESUMEN
Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression of P. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containing P. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivalis colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.
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Infecciones por Bacteroidaceae/microbiología , Vasos Coronarios/microbiología , Endotelio Vascular/microbiología , Fagosomas/microbiología , Porphyromonas gingivalis/patogenicidad , Autofagia , Línea Celular , Vasos Coronarios/ultraestructura , Endocitosis , Endotelio Vascular/ultraestructura , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Fagosomas/ultraestructura , Porphyromonas gingivalis/fisiologíaRESUMEN
Vacuolar H(+)-ATPases (V-ATPases) are essential for acidification of intracellular compartments and for proton secretion from the plasma membrane in kidney epithelial cells and osteoclasts. The cellular proteins that regulate V-ATPases remain largely unknown. A screen for proteins that bind the V-ATPase E subunit using the yeast two-hybrid assay identified the cDNA clone coded for aldolase, an enzyme of the glycolytic pathway. The interaction between E subunit and aldolase was confirmed in vitro by precipitation assays using E subunit-glutathione S-transferase chimeric fusion proteins and metabolically labeled aldolase. Aldolase was isolated associated with intact V-ATPase from bovine kidney microsomes and osteoclast-containing mouse marrow cultures in co-immunoprecipitation studies performed using an anti-E subunit monoclonal antibody. The interaction was not affected by incubation with aldolase substrates or products. In immunocytochemical assays, aldolase was found to colocalize with V-ATPase in the renal proximal tubule. In osteoclasts, the aldolase-V-ATPase complex appeared to undergo a subcellular redistribution from perinuclear compartments to the ruffled membranes following activation of resorption. In yeast cells deficient in aldolase, the peripheral V(1) domain of V-ATPase was found to dissociate from the integral membrane V(0) domain, indicating direct coupling of glycolysis to the proton pump. The direct binding interaction between V-ATPase and aldolase may be a new mechanism for the regulation of the V-ATPase and may underlie the proximal tubule acidification defect in hereditary fructose intolerance.
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Fructosa-Bifosfato Aldolasa/metabolismo , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón Vacuolares , Animales , Sitios de Unión , Bovinos , Membrana Celular/enzimología , Núcleo Celular , ADN Complementario/metabolismo , Fructosa/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Glucosa-6-Fosfato Isomerasa/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Riñón/metabolismo , Microsomas/metabolismo , Mutación , Osteoclastos/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Protones , Técnicas del Sistema de Dos HíbridosRESUMEN
To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.
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Citoplasma/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Peroxisomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Vacuolas/metabolismo , Proteínas Relacionadas con la Autofagia , Membrana Celular/metabolismo , Supervivencia Celular , Clonación Molecular , Escherichia coli/metabolismo , Genotipo , Proteínas de la Membrana/química , Microscopía Fluorescente , Nitrógeno/metabolismo , Fagocitosis , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Fracciones Subcelulares , Factores de Tiempo , TransfecciónRESUMEN
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.
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Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Transducción de Señal/fisiología , Vesículas Transportadoras/metabolismo , Vacuolas/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Western Blotting , Proteínas Portadoras/genética , Fraccionamiento Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Glucosa/metabolismo , Humanos , Microscopía Fluorescente , Peroxisomas/metabolismo , Pichia/genética , Pichia/metabolismo , Pichia/ultraestructura , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructuraRESUMEN
Aldolase B is an abundant cytosolic protein found in all eukaryotic cells. Like many glycolytic enzymes, this protein was sequestered into lysosomes for degradation during nutrient starvation. We report here that the degradation of recombinant aldolase B was enhanced two-fold when rat and human hepatoma cells were starved for amino acid and serum. In addition, starvation-induced degradation of aldolase B was inhibited by chloroquine, an inhibitor of lysosomal proteinases and by 3-methyladenine, an inhibitor of autophagy. Aldolase B has three lysosomal targeting motifs (Q(12)KKEL, Q(58)FREL, and IKLDQ(111)) that have been proposed to interact with hsc73 thereby initiating its transport into lysosomes. In this study, we have mutated the essential glutamine residues in each of these hsc73-binding motifs in order to evaluate their roles in the lysosomal degradation of aldolase B during starvation. We have found that when glutamines 12 or 58 are mutated to asparagines enhanced degradation of aldolase B proceeded normally. However, when glutamine 111 was mutated to an asparagine or a threonine, starvation-induced degradation was completely suppressed. These mutations did not appear to alter the tertiary structure of aldolase B since enzymatic activity was not affected. Our results suggest that starvation-induced lysosomal degradation of aldolase B requires both autophagy and glutamine 111. We discuss the possible roles for autophagy and hsc73-mediated transport in the lysosomal sequestration of aldolase B.
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Aminoácidos/deficiencia , Fructosa-Bifosfato Aldolasa/metabolismo , Glutamina/fisiología , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico/fisiología , Lisosomas/fisiología , Secuencias de Aminoácidos , Animales , Autofagia , Secuencia de Bases , Carcinoma Hepatocelular , Medio de Cultivo Libre de Suero/farmacología , Epítopos/genética , Epítopos/aislamiento & purificación , Epítopos/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Proteínas del Choque Térmico HSC70 , Humanos , Lisosomas/efectos de los fármacos , Chaperonas Moleculares/fisiología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Transporte de Proteínas , Ratas , Alineación de Secuencia , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G(0) program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis.
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Transporte Activo de Núcleo Celular/fisiología , Autofagia/fisiología , Fosfatidilinositol 3-Quinasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Vacuolas/enzimología , Proteínas Adaptadoras Transductoras de Señales , Aminopeptidasas/metabolismo , Antifúngicos/farmacología , Autofagia/efectos de los fármacos , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Proteínas de Ciclo Celular , Cicloheximida/farmacología , Citoplasma/enzimología , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/fisiología , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación/fisiología , Nitrógeno/farmacología , Fenotipo , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Sirolimus/farmacología , Proteínas de Transporte VesicularRESUMEN
There is an emerging paradigm shift from coronary heart disease having a purely hereditary and nutritional causation to possibly having an infectious etiology. Recent epidemiological studies have shown a correlation between periodontal disease and coronary heart disease. However, to date, there is minimal information as to the possible disease mechanisms of this association. It is our hypothesis that invasion of the coronary artery cells by oral bacteria may start and/or exacerbate the inflammatory response in atherosclerosis. Since a few periodontal pathogens have been reported to invade oral epithelial tissues, we tested the ability of three putative periodontal pathogens-Eikenella corrodens, Porphyromonas gingivalis, and Prevotella intermedia-to invade human coronary artery endothelial cells and coronary artery smooth muscle cells. In this study we demonstrate by an antibiotic protection assay and electron microscopy that specific species and strains invade coronary artery cells at a significant level. Actin polymerization and eukaryotic protein synthesis in metabolically active cells were required since the corresponding inhibitors nearly abrogated invasion. Many intracellular P. gingivalis organisms were seen to be present in multimembranous vacuoles resembling autophagosomes by morphological analysis. This is the first report of oral microorganisms invading human primary cell cultures of the vasculature.
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Vasos Coronarios/microbiología , Eikenella corrodens/fisiología , Porphyromonas gingivalis/fisiología , Prevotella intermedia/fisiología , Vasos Coronarios/ultraestructura , Citocalasina D/farmacología , Endotelio Vascular/microbiología , Endotelio Vascular/ultraestructura , Humanos , Células KB , Microscopía Electrónica , TemperaturaRESUMEN
Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.
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Autofagia/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microcuerpos/metabolismo , Pichia/metabolismo , Proteínas de Saccharomyces cerevisiae , Adaptación Fisiológica , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteína 7 Relacionada con la Autofagia , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Glucosa/metabolismo , Humanos , Ligasas/genética , Datos de Secuencia Molecular , Mutación , Pichia/genética , Homología de Secuencia de Aminoácido , Enzimas Activadoras de Ubiquitina , Ubiquitina-Proteína LigasasRESUMEN
We have previously shown that stress-induced protein degradation requires a functional ubiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of ubiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress by enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed by pactamycin, cycloheximide, and puromycin. We observed an accumulation of ubiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein ubiquitination was not affected by cycloheximide, indicating that protein synthesis was not required. The ubiquitinated proteins were localized to the cytosol and subcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the ubiquitinated proteins into autolysosomes was dramatically reduced by 3-methyladenine, an inhibitor of autophagy. The evidence suggests that ubiquitinated proteins are sequestered by autophagy for degradation. We next set out to identify those primary ubiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antibodies were prepared against these proteins that had been immunopurified from rat liver lysosomes. The antibodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences obtained from two cyanogen bromide fragments of this 40 kDa protein were shown to be identical to sequences in liver fructose1,6-bisphosphate aldolase B. Anti-Ub68 antibodies recognized purified aldolase A and aldolase B. Conversely, antibodies prepared against aldolase B recognized the 40 kDa aldolase as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of aldolase B was enhanced during amino acid and serum starvation. This degradation was suppressed by chloroquine and 3-methyladenine, suggesting that aldolase B was being degraded within autolysosomes. We propose that aldolase B is ubiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Lisosomas/enzimología , Inanición/metabolismo , Ubiquitinas/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Aminoácidos/farmacología , Animales , Antimaláricos/farmacología , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacología , Carcinoma Hepatocelular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cloroquina/farmacología , Citosol/enzimología , Fructosa-Bifosfato Aldolasa/genética , Riñón/citología , Hígado/metabolismo , Datos de Secuencia Molecular , Ratas , Células Tumorales CultivadasRESUMEN
We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells. When P. pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized. During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy. We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes. In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole. In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1. Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115. Third, a PFK1 knockout (delta pfk1) is unable to degrade alcohol oxidase during glucose adaptation. As observed in gsa1, the peroxisomes in delta pfk1 remain outside the vacuole during adaptation. Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e. signaling, selection, or sequestration). However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase. The inability of delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation. We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.
Asunto(s)
Autofagia/fisiología , Glucosa/farmacología , Fosfofructoquinasa-1/metabolismo , Pichia/fisiología , Secuencia de Aminoácidos , Animales , Autofagia/efectos de los fármacos , Secuencia de Bases , Citosol/enzimología , Genes Fúngicos , Humanos , Sustancias Macromoleculares , Mamíferos , Metanol/metabolismo , Microcuerpos/enzimología , Datos de Secuencia Molecular , Mutación , Fosfofructoquinasa-1/biosíntesis , Fosfofructoquinasa-1/química , Pichia/efectos de los fármacos , Pichia/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transformación GenéticaRESUMEN
The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroautophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.