RESUMEN
The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.
Asunto(s)
Caperuzas de ARN , ARN Viral , SARS-CoV-2 , Proteínas Virales , Antivirales , COVID-19/virología , Dominio Catalítico , Guanosina Difosfato/metabolismo , Humanos , Metiltransferasas/metabolismo , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Dominios Proteicos , Caperuzas de ARN/química , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , SARS-CoV-2/enzimología , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.
Asunto(s)
AMP Cíclico , Proteínas de Drosophila , Drosophila melanogaster , Estrés del Retículo Endoplásmico , Nucleotidiltransferasas , Estrés Fisiológico , Respuesta de Proteína Desplegada , Animales , Ratones , Alelos , AMP Cíclico/metabolismo , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Nucleotidiltransferasas/deficiencia , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Páncreas/efectos de los fármacos , Páncreas/enzimología , Páncreas/metabolismo , Páncreas/fisiopatología , Estrés Fisiológico/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Regulación de la Expresión Génica , Fosfatos , Transporte Biológico , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Fosfatos/metabolismoRESUMEN
The deleterious effects of statins on skeletal muscle are well known, but the mechanism associated with these effects remains unresolved. Statins are associated with mitochondrial damage, which may contribute to muscle myopathy. Here we demonstrate that simvastatin induces mitophagy in skeletal muscle cells and hypothesized that attenuating this process by silencing the mitophagy adapter p62/sequestosome-1 (SQSTM1) might mitigate myotoxicity. Surprisingly, silencing p62/SQSTM1 in differentiated C2C12 muscle cells exacerbated rather than attenuated myotoxicity. This inhibition of mitophagy in the face of statin challenge correlated with increased release of cytochrome c to the cytosol, activation of caspase-3, and lactate dehydrogenase (LDH) release. Correspondingly, targeted knockdown of Parkin, a canonical E3 ubiquitin ligase important for mitophagy, mirrored the effects of p62/SQSTM1 silencing. To corroborate these findings in vivo, we treated Parkin knockout mice with simvastatin for 2 wk. In line with our findings in vitro, these mitophagy-compromised mice displayed reduced spontaneous activity, loss of grip strength, and increased circulating levels of muscle damage marker LDH. Our findings demonstrate that mitophagy is an important mechanism to resist statin-induced skeletal muscle damage.-Ramesh, M., Campos, J. C., Lee, P., Song, Y., Hernandez, G., Sin, J., Tucker, K. C., Saadaeijahromi, H., Gurney, M., Ferreira, J. C. B., Andres, A. M. Mitophagy protects against statin-mediated skeletal muscle toxicity.
Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Mitofagia/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Simvastatina/farmacología , Animales , Caspasa 3/metabolismo , Línea Celular , Citocromos c/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Ratones Noqueados , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Interferencia de ARN , Proteína Sequestosoma-1/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Sigma (σ) receptors have generated a great deal of interest due to their possible role in psychosis, neuroprotection, and various other behaviors including addictive processes. Sigma receptors have been located in brain areas involved in motor functions, including the dopaminergic projections from the substantia nigra to the striatum. Evidence suggests that one of their major roles might be to regulate the activity of the glutamatergic system via the N-methyl-D-aspartate receptor. The sigma receptor agonist 1,3-di-o-tolyl-guanidine (DTG) was found to increase dopamine release in the striatum, nucleus accumbens, and prefrontal cortex, in a dose-dependent manner, after central as well as peripheral administration, suggesting a modulatory role of these receptors on the dopaminergic system. The present study examines whether chronic administration of the DTG sigma agonist induces neuromorphological and behavioral changes in neonatal ventral hippocampal lesioned (nVHL) rats as a neurodevelopmental model of schizophrenia. The results show that the DTG administration reduces the hyperlocomotor activity in nVHL rats and reverses the neuronal hypotrophy generated in nVHL rats in the prefrontal cortex, amygdala, and nucleus accumbens. Our results demonstrate that DTG, a sigma-1 receptor agonist, reverses some of the behavioral and neuromorphological effects of nVHL on the rat and supports the possibility that DTG may have beneficial effects in the management of symptoms of schizophrenia.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Conducta Animal/efectos de los fármacos , Lesiones Encefálicas , Guanidinas/uso terapéutico , Hipocampo/patología , Análisis de Varianza , Animales , Animales Recién Nacidos , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Conducta Exploratoria/efectos de los fármacos , Hipocampo/ultraestructura , Masculino , Actividad Motora/efectos de los fármacos , Inhibición Prepulso/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reflejo de Sobresalto/efectos de los fármacos , Tinción con Nitrato de PlataRESUMEN
Cancer is the second leading cause of death worldwide, despite the many treatments available, cancer patients face side effects that reduce their quality of life. Therefore, there is a need to develop novel strategies to increase the efficacy of treatments. In this study, gold nanoparticles obtained by green synthesis with Coffea arabica green bean extract were loaded with Doxorubicin, (a highly effective but non-specific drug) by direct interaction and using commercial organic ligands that allow colloidal dispersion at physiological and tumor pH. Conjugation of these components resulted in stable nanohybrids at physiological pH and a tumor pH release dependent, with a particle size less than 40 nm despite having the ligands and Doxorubicin loaded on their surface, which gave them greater specificity and cytotoxicity in H69 tumor cells.
RESUMEN
The use of immunomodulatory and metabolic modulating drugs has been considered a better strategy to improve the efficacy of conventional treatments against pathogens and metabolic diseases. L-carnitine is relevant in fatty acid metabolism and energy production by ß-oxidation, but it also has a beneficial therapeutic immunomodulatory effect. The ß-hydroxy-γ-aminophosphonate (ß-HPC) was developed, synthesized and studied in different pathologies as a more soluble and stable analog than L-carnitine, which has been studied in bacterial physiology and metabolism; therefore, we set out to investigate the direct effect of ß-HPC on the metabolism of N. brasiliensis, which causes actinomycetoma in Mexico and is underdiagnosed. To analyze the effect of ß-HPC on the metabolic capacity of the bacterium for the hydrolysis of substrate casein, L-tyrosine, egg yolk, and tween 80, Fourier transform infrared spectroscopy (FT-IR) was employed. It was found that ß-HPC increases the metabolic activity of N. brasiliensis associated with increased growth and increased hydrolysis of the substrates tested. By the effect of ß-HPC, it was observed that, in the hydrolysis of L-tyrosine, the aromatic ring and functional groups were degraded. At 1515 cm-1, any distinctive signal or peak for this amino acid was missing, almost disappearing at 839, 720, 647, and 550 cm-1. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, and CO. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, COO, and P = O signals, characteristic of amino acids, in addition to the increase of the amide I and II bands. In Tween 80 the H-C = and C = C signals disappear and the ether signals are concentrated, it was distinguished by the intense band at 1100 cm-1. Egg yolk showed a large accumulation of phosphate groups at 1071 cm-1, where phosvitin is located. FT-IR has served to demonstrate that ß-HPC is a hydrolysis enhancer. Furthermore, by obtaining the spectrum of N. brasiliensis, we intend to use it as a quick comparison tool with other spectra related to actinobacteria. Eventually, FT-IR may serve as a species identification option.
RESUMEN
The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2'-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.
RESUMEN
Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity.
Asunto(s)
Clonación Molecular/métodos , Histonas/biosíntesis , Histonas/aislamiento & purificación , Animales , Proteínas Aviares/biosíntesis , Proteínas Aviares/genética , Proteínas Aviares/aislamiento & purificación , Pollos , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/genética , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , SolubilidadRESUMEN
The exocrine pancreas expresses the highest concentrations of fibroblast growth factor 21 (FGF21) in the body, where it maintains acinar cell proteostasis. Here, we showed in both mice and humans that acute and chronic pancreatitis is associated with a loss of FGF21 expression due to activation of the integrated stress response (ISR) pathway. Mechanistically, we found that activation of the ISR in cultured acinar cells and mouse pancreata induced the expression of ATF3, a transcriptional repressor that directly bound to specific sites on the Fgf21 promoter and resulted in loss of FGF21 expression. These ATF3 binding sites are conserved in the human FGF21 promoter. Consistent with the mouse studies, we also observed the reciprocal expression of ATF3 and FGF21 in the pancreata of human patients with pancreatitis. Using three different mouse models of pancreatitis, we showed that pharmacologic replacement of FGF21 mitigated the ISR and resolved pancreatitis. Likewise, inhibition of the ISR with an inhibitor of the PKR-like endoplasmic reticulum kinase (PERK) also restored FGF21 expression and alleviated pancreatitis. These findings highlight the importance of FGF21 in preserving exocrine pancreas function and suggest its therapeutic use for prevention and treatment of pancreatitis.
Asunto(s)
Factores de Crecimiento de Fibroblastos/deficiencia , Pancreatitis/terapia , Células Acinares/metabolismo , Células Acinares/patología , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 4 , Animales , Secuencia de Bases , Regulación hacia Abajo , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Humanos , Proteínas Klotho , Ratones Noqueados , Páncreas Exocrino/patología , Pancreatitis/genética , Pancreatitis/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/metabolismoRESUMEN
In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of approximately 21 bp PSEA and approximately 8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5' half of the PSEA is well-conserved, but the 3' half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3' half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.
Asunto(s)
Evolución Molecular , Genes de Insecto , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , Animales , Anopheles/genética , Secuencia de Bases , Abejas/genética , Bombyx/genética , Secuencia Conservada , Drosophila/genética , Drosophila melanogaster/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Alcohol and ketogenic diets increase water consumption. Here, we show that the hormone FGF21 is required for this drinking response in mice. Circulating levels of FGF21 are increased by alcohol consumption in humans and by both alcohol and ketogenic diets in mice. Pharmacologic administration of FGF21 stimulates water drinking behavior in mice within 2 hr. Concordantly, mice lacking FGF21 fail to increase water intake in response to either alcohol or a ketogenic diet. The effect of FGF21 on drinking is mediated in part by SIM1-positive neurons of the hypothalamus and is inhibited by ß-adrenergic receptor antagonists. Given that FGF21 also is known to suppress alcohol intake in favor of pure water, this work identifies FGF21 as a fundamental neurotropic hormone that governs water balance in response to specific nutrient stresses that can cause dehydration.
Asunto(s)
Consumo de Bebidas Alcohólicas/efectos adversos , Dieta Cetogénica/efectos adversos , Ingestión de Líquidos/fisiología , Factores de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/fisiología , Antagonistas Adrenérgicos beta/administración & dosificación , Adulto , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ingestión de Líquidos/efectos de los fármacos , Femenino , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/genética , Voluntarios Sanos , Humanos , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
The metabolic stress hormone FGF21 is highly expressed in exocrine pancreas, where its levels are increased by refeeding and chemically induced pancreatitis. However, its function in the exocrine pancreas remains unknown. Here, we show that FGF21 stimulates digestive enzyme secretion from pancreatic acinar cells through an autocrine/paracrine mechanism that requires signaling through a tyrosine kinase receptor complex composed of an FGF receptor and ß-Klotho. Mice lacking FGF21 accumulate zymogen granules and are susceptible to pancreatic ER stress, an effect that is reversed by administration of recombinant FGF21. Mice carrying an acinar cell-specific deletion of ß-Klotho also accumulate zymogen granules but are refractory to FGF21-stimulated secretion. Like the classical post-prandial secretagogue, cholecystokinin (CCK), FGF21 triggers intracellular calcium release via PLC-IP3R signaling. However, unlike CCK, FGF21 does not induce protein synthesis, thereby preventing protein accumulation. Thus, pancreatic FGF21 is a digestive enzyme secretagogue whose physiologic function is to maintain acinar cell proteostasis.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Páncreas Exocrino/metabolismo , Animales , Comunicación Autocrina , Calcio/metabolismo , Digestión , Estrés del Retículo Endoplásmico , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Espacio Intracelular/metabolismo , Masculino , Ratones Noqueados , Páncreas Exocrino/enzimología , Comunicación Paracrina , Fosfolipasa C gamma/metabolismo , Biosíntesis de Proteínas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de SeñalRESUMEN
Fibroblast growth factor 21 (FGF21) is a hormone induced by various metabolic stresses, including ketogenic and high-carbohydrate diets, that regulates energy homeostasis. In humans, SNPs in and around the FGF21 gene have been associated with macronutrient preference, including carbohydrate, fat, and protein intake. Here we show that FGF21 administration markedly reduces sweet and alcohol preference in mice and sweet preference in cynomolgus monkeys. In mice, these effects require the FGF21 co-receptor ß-Klotho in the central nervous system and correlate with reductions in dopamine concentrations in the nucleus accumbens. Since analogs of FGF21 are currently undergoing clinical evaluation for the treatment of obesity and type 2 diabetes, our findings raise the possibility that FGF21 administration could affect nutrient preference and other reward behaviors in humans.
Asunto(s)
Alcoholes/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Preferencias Alimentarias/efectos de los fármacos , Gusto/efectos de los fármacos , Animales , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Dopamina/metabolismo , Haplorrinos , Humanos , Masculino , Ratones Endogámicos C57BL , Sacarina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
AIMS: We have shown that autophagy and mitophagy are required for preconditioning. While statin's cardioprotective effects are well known, the role of autophagy/mitophagy in statin-mediated cardioprotection is not. In this study, we used HL-1 cardiomyocytes and mice subjected to ischemia/reperfusion to elucidate the mechanism of statin-mediated cardioprotection. RESULTS: HL-1 cardiomyocytes exposed to simvastatin for 24 h exhibited diminished protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling, increased activation of unc-51-like kinase 1, and upregulation of autophagy and mitophagy. Similar findings were obtained in hearts of mice given simvastatin. Mevalonate abolished simvastatin's effects on Akt/mTOR signaling and autophagy induction in HL-1 cells, indicating that the effects are mediated through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Simvastatin-treated HL-1 cells exhibited mitochondrial translocation of Parkin and p62/SQSTM1, fission, and mitophagy. Because Parkin is required for mitophagy and is expressed in heart, we investigated the effect of simvastatin on infarct size in Parkin knockout mice. Simvastatin reduced infarct size in wild-type mice but showed no benefit in Parkin knockout mice. Inhibition of HMG-CoA reductase limits mevalonate availability for both cholesterol and coenzyme Q10 (CoQ) biosynthesis. CoQ supplementation had no effect on statin-induced Akt/mTOR dephosphorylation or macroautophagy in HL-1 cells, but it potently blocked mitophagy. Importantly, CoQ supplementation abolished statin-mediated cardioprotection in vivo. INNOVATION AND CONCLUSION: Acute simvastatin treatment suppresses mTOR signaling and triggers Parkin-dependent mitophagy, the latter which is required for cardioprotection. Coadministration of CoQ with simvastatin impairs mitophagy and cardioprotection. These results raise the concern that CoQ may interfere with anti-ischemic benefits of statins mediated through stimulation of mitophagy.
Asunto(s)
Cardiotónicos/administración & dosificación , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Simvastatina/administración & dosificación , Animales , Autofagia/efectos de los fármacos , Homólogo de la Proteína 1 Relacionada con la Autofagia , Línea Celular , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Ratones , Mitofagia/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Serina-Treonina Quinasas/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-akt/genética , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/biosíntesis , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial-targeting sequence (coined "MitoTimer") and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and production was induced with doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Dox addition for as little as 1 h was sufficient to induce MitoTimer expression within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent transition from green to red over 48 h and was amenable to analysis by fluorescence microscopy and flow cytometry of whole cells or isolated mitochondria. A second Dox administration 48 h after the initial induction resulted in a second round of expression of green MitoTimer. The extent of new protein incorporation during a second pulse was increased by administration of a mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Recambio Mitocondrial , Proteínas Mutantes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Citometría de Flujo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Factores de Tiempo , Proteína Fluorescente RojaRESUMEN
Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas de Choque Térmico/metabolismo , Precondicionamiento Isquémico Miocárdico , Mitocondrias/metabolismo , Mitocondrias/patología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Polaridad Celular , Ratones , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Transporte de Proteínas , Daño por Reperfusión/prevención & control , Proteína Sequestosoma-1 , Estrés FisiológicoRESUMEN
The Stanford HIVdb is implementing three applications to address the issue of HIV drug resistance. Knowledge integration of the applications is desirable. How best to integrate the software applications is the main question. We have pursued two possibilities for accomplishing knowledge integration. The most feasible method appears to use mapping ontologies. Future work is focused on defining a set of mapping relations to aid in the knowledge integration.
Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Programas Informáticos , Integración de Sistemas , Investigación Biomédica , HumanosRESUMEN
Syntheses of pyrazoles featuring a functionalized side chain attached to carbon 3 and varying alkyl and aryl substituents attached to carbon 5 are presented. Installation of R = methyl, isopropyl, tert-butyl, adamantyl, or phenyl groups at C5 is reported here, starting by coupling protected alkynols with acid chlorides RCOCl, forming alkynyl ketones, which are reacted with hydrazine to form the pyrazole nucleus. Alcohol deprotection and conversion to a chloride gave 5-substituted 3-(chloromethyl)- or 3-(2-chloroethyl)pyrazoles. This sequence can be done within 2 d on a 30 g scale in excellent overall yield. Through nucleophilic substitution reactions, the chlorides are useful precursors to other polyfunctional pyrazoles. In the work here, derivatives with side chains LCH(2)- and LCH(2)CH(2)- at C3 (L = thioether or phosphine) were made as ligands. The significance of the ligands made here is that by placing a ligating side chain on a ring carbon (C3), rather than on a ring nitrogen, the ring nitrogen not bound to the metal and its attached proton will be available for hydrogen bonding, depending on the steric environment created by R at C5.
RESUMEN
Inter- and intramolecular hydrogen bonding of an N-H group in pyrazole complexes was studied using ligands with two different groups at pyrazole C-3 and C-5. At C-5, groups such as methyl, i-propyl, phenyl, or tert-butyl were present. At C-3, side chains L-CH(2)- and L-CH(2)CH(2)- (L = thioether or phosphine) ensured formation of chelates to a cis-dichloropalladium(II) fragment through side-chain atom L and the pyrazole nitrogen closest to the side chain. The significance of the ligands is that by placing a ligating side chain on a ring carbon (C-3), rather than on a ring nitrogen, the ring nitrogen not bound to the metal and its attached proton are available for hydrogen bonding. As desired, seven chelate complexes examined by X-ray diffraction all showed intramolecular hydrogen bonding between the pyrazole N-H and a chloride ligand in the cis position. In addition, however, intermolecular hydrogen bonding could be controlled by the substituent at C-5: complexes with either a methyl at C-5 or no substituent there showed significant intermolecular hydrogen bonding interactions, which were completely avoided by placing a tert-butyl group at C-5. The acidity of two complexes in acetonitrile solutions was estimated to be closer to that of pyridinium ion than those of imidazolium or triethylammonium ions.