RESUMEN
Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Luciferasas , Proteínas de Choque Térmico/química , Escherichia coli/metabolismo , Pliegue de Proteína , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Escherichia coli/química , Proteínas Bacterianas/química , Proteínas HSP70 de Choque Térmico/química , Chaperonas Moleculares/químicaRESUMEN
AMP kinase is a heterotrimeric serine/threonine protein kinase that regulates a number of metabolic processes, including lipid biosynthesis and metabolism. AMP kinase activity is regulated by phosphorylation, and the kinases involved have been uncovered. The particular phosphatases counteracting these kinases remain elusive. Here we discovered that the protein phosphatase 2A heterotrimer, PP2A(Ppp2r2d), regulates the phosphorylation state of AMP kinase by dephosphorylating Thr-172, a residue that activates kinase activity when phosphorylated. Co-immunoprecipitation and co-localization studies indicated that PP2A(Ppp2r2d) directly interacted with AMP kinase. PP2A(Ppp2r2d) dephosphorylated Thr-172 in rat aortic and human vascular smooth muscle cells. A positive correlation existed between decreased phosphorylation, decreased acetyl-CoA carboxylase Acc1 phosphorylation, and sterol response element-binding protein 1c-dependent gene expression. PP2A(Ppp2r2d) protein expression was up-regulated in the aortas of mice fed a high fat diet, and the increased expression correlated with increased blood lipid levels. Finally, we found that the aortas of mice fed a high fat diet had decreased AMP kinase Thr-172 phosphorylation, and contained an Ampk-PP2A(Ppp2r2d) complex. Thus, PP2A(Ppp2r2d) may antagonize the aortic AMP kinase activity necessary for maintaining normal aortic lipid metabolism. Inhibiting PP2A(Ppp2r2d) or activating AMP kinase represents a potential pharmacological treatment for many lipid-related diseases.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/química , Animales , Aorta/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Línea Celular , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversos , Inhibidores Enzimáticos/farmacología , Humanos , Metabolismo de los Lípidos , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Ocadaico/farmacología , Fosforilación , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de Proteína , ARN Interferente Pequeño/genética , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake.
Asunto(s)
LDL-Colesterol/metabolismo , Proteína Fosfatasa 2/metabolismo , Elementos de Respuesta , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Transporte Activo de Núcleo Celular , LDL-Colesterol/deficiencia , Células HEK293 , Células Hep G2 , Humanos , Unión Proteica , Proteína Fosfatasa 2/genética , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function.
Asunto(s)
Colesterol/biosíntesis , Lipoproteínas LDL/metabolismo , MicroARNs/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Colesterol/sangre , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Insulina/farmacología , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Nucleares Huérfanos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Receptores de LDL/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Saccharomyces cerevisiae ergosterol biosynthesis, like cholesterol biosynthesis in mammals, is regulated at the transcriptional level by a sterol feedback mechanism. Yeast studies defined a 7-bp consensus sterol-response element (SRE) common to genes involved in sterol biosynthesis and two transcription factors, Upc2 and Ecm22, which direct transcription of sterol biosynthetic genes. The 7-bp consensus SRE is identical to the anaerobic response element, AR1c. Data indicate that Upc2 and Ecm22 function through binding to this SRE site. We now show that it is two novel anaerobic AR1b elements in the UPC2 promoter that direct global ERG gene expression in response to a block in de novo ergosterol biosynthesis, brought about by antifungal drug treatment. The AR1b elements are absolutely required for auto-induction of UPC2 gene expression and protein and require Upc2 and Ecm22 for function. We further demonstrate the direct binding of recombinant expressed S. cerevisiae ScUpc2 and pathogenic Candida albicans CaUpc2 and Candida glabrata CgUpc2 to AR1b and SRE/AR1c elements. Recombinant endogenous promoter studies show that the UPC2 anaerobic AR1b elements act in trans to regulate ergosterol gene expression. Our results indicate that Upc2 must occupy UPC2 AR1b elements in order for ERG gene expression induction to take place. Thus, the two UPC2-AR1b elements drive expression of all ERG genes necessary for maintaining normal antifungal susceptibility, as wild type cells lacking these elements have increased susceptibility to azole antifungal drugs. Therefore, targeting these specific sites for antifungal therapy represents a novel approach to treat systemic fungal infections.
Asunto(s)
Genes Fúngicos , Elementos de Respuesta , Esteroles/metabolismo , Aerobiosis , Anaerobiosis , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Candida glabrata/efectos de los fármacos , Candida glabrata/genética , Candida glabrata/metabolismo , Ergosterol/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Lovastatina/farmacología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4(+) T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Productos del Gen tax/genética , MicroARNs/genética , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Retroviridae/genética , Linfocitos T/metabolismo , Southern Blotting , Western Blotting , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Luciferasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Secuencias Repetidas Terminales/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.
Asunto(s)
Candida albicans/genética , Candida albicans/patogenicidad , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Esteroles/metabolismo , Animales , Antifúngicos , Resistencia a Múltiples Medicamentos/genética , Regulación Fúngica de la Expresión Génica , Metabolismo de los Lípidos/genética , Ratones , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
CCAAT/enhancer-binding protein (C/EBP) basic region/leucine zipper (bZIP) transcription factors have been shown to form heterodimers with cAMP-responsive element binding protein 2 (CREB-2), a transcription factor involved in regulating basal and Tax-mediated transactivation of the human T cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR). In cells of the monocyte-macrophage lineage (proposed to play a role in HTLV-1 pathogenesis as an accessory target cell), several members of the C/EBP family are expressed at high levels and may have functional impact on both basal and Tax-mediated transactivation of the HTLV-1 LTR. Basal activation of the HTLV-1 LTR was enhanced by overexpression of C/EBPbeta, C/EBPdelta, or C/EBPepsilon, whereas transactivation of the LTR by Tax was inhibited by overexpression of C/EBPalpha and C/EBPbeta. Inhibition of Tax-mediated transactivation of the HTLV-1 LTR was co-activator-independent, did not require C/EBP binding to the Tax-responsive elements, and may involve heterodimerization with CREB factors.