RESUMEN
Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
Asunto(s)
Lisina-ARNt Ligasa , Mycobacterium tuberculosis , Tuberculosis , Animales , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/farmacología , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológicoRESUMEN
Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in Leishmania major [H. Guo et al., J. Biol. Chem. 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative "plasmid segregation" assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null ∆fut1- background, established that FUT1 is essential. Similarly, "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes, respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly, a single rare ∆fut1- segregant (∆fut1s ) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 reexpression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 [G. Bandini et al., bioRxiv, https://www.biorxiv.org/content/10.1101/726117v2 (2021)] joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting, and their essentiality for normal growth and mitochondrial function renders it an attractive target for chemotherapy of these serious human pathogens.
Asunto(s)
Fucosiltransferasas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Leishmania major/metabolismo , Mitocondrias/enzimología , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Medios de Cultivo , Fucosiltransferasas/genética , Mutación , Plásmidos , Transporte de Proteínas , Proteínas Protozoarias/genética , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei. Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: ß-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galß1,3GlcNAcß1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.
Asunto(s)
Fucosiltransferasas/metabolismo , Mitocondrias/enzimología , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Fucosiltransferasas/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Filogenia , Trypanosoma brucei brucei/enzimología , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Carboxiliasas/antagonistas & inhibidores , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Péptido Sintasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Carboxiliasas/genética , Carboxiliasas/metabolismo , Carboxiliasas/ultraestructura , Coenzima A/biosíntesis , Cristalografía por Rayos X , Pruebas de Enzimas , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Péptido Sintasas/ultraestructura , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Ratones SCID , Proteínas Protozoarias/metabolismoRESUMEN
Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.
Asunto(s)
Fucosa/metabolismo , Glicoconjugados/metabolismo , Guanosina Difosfato Fucosa/biosíntesis , Plasmodium falciparum/metabolismo , Vías Biosintéticas/genética , Genoma de Protozoos/genética , Hidroliasas/genética , Hidroliasas/metabolismo , Microscopía Fluorescente , Mutación , Fenotipo , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.
Asunto(s)
Glucosa/metabolismo , Glutamina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/fisiología , Uridina Difosfato N-Acetilglucosamina/metabolismo , Animales , Proliferación Celular/genética , Autorrenovación de las Células/genética , Transformación Celular Neoplásica/genética , Células Clonales , Femenino , Activación de Linfocitos/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Receptores Notch/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pntd.0004205.].
RESUMEN
Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.
Asunto(s)
Leishmania major/crecimiento & desarrollo , Leishmania major/metabolismo , Uridina Difosfato Galactosa/deficiencia , Uridina Difosfato Glucosa/deficiencia , Eliminación de Gen , Regulación de la Expresión Génica , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection.
Asunto(s)
Galactosa/metabolismo , Glicoconjugados/metabolismo , Leishmania major/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Eliminación de Gen , Leishmania major/genética , Leishmania major/crecimiento & desarrollo , Leishmania major/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/genéticaRESUMEN
Uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central position in carbohydrate metabolism in all kingdoms of life, since its product uridine diphosphate-glucose (UDP-glucose) is essential in a number of anabolic and catabolic pathways and is a precursor for other sugar nucleotides. Its significance as a virulence factor in protists and bacteria has given momentum to the search for species-specific inhibitors. These attempts are, however, hampered by high structural conservation of the active site architecture. A feature that discriminates UGPs of different species is the quaternary organization. While UGPs in protists are monomers, di- and tetrameric forms exist in bacteria, and crystal structures obtained for the enzyme from yeast and human identified octameric UGPs. These octamers are formed by contacts between highly conserved amino acids in the C-terminal ß-helix. Still under debate is the question whether octamerization is required for the functionality of the human enzyme. Here, we used single amino acid replacements in the C-terminal ß-helix to interrogate the impact of highly conserved residues on octamer formation and functional activity of human UGP (hUGP). Replacements were guided by the sequence of Arabidopsis thaliana UGP, known to be active as a monomer. Correlating the data obtained in blue native PAGE, size exclusion chromatography and enzymatic activity testing, we prove that the octamer is the active enzyme form. This new insight into structure-function relationships in hUGP does not only improve the understanding of the catalysis of this important enzyme, but in addition broadens the basis for studies aimed at designing drugs that selectively inhibit UGPs from pathogens.
Asunto(s)
Dominio Catalítico , Multimerización de Proteína , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Arabidopsis/enzimología , Secuencia Conservada , Humanos , Mutación , UTP-Glucosa-1-Fosfato Uridililtransferasa/genéticaRESUMEN
Nucleotide sugars and the enzymes that are responsible for their synthesis are indispensable for the production of complex carbohydrates and, thus, for elaboration of a protective cellular coat for many organisms such as the protozoan parasite Leishmania. These activated sugars are synthesized de novo or derived from salvaged monosaccharides. In addition to UDP-glucose (UDP-Glc) pyrophosphorylase, which catalyzes the formation of UDP-Glc from substrates UTP and glucose-1-phosphate, Leishmania major and plants express a UDP-sugar pyrophosphorylase (USP) that exhibits broad substrate specificity in vitro. The enzyme, likely involved in monosaccharide salvage, preferentially generates UDP-Glc and UDP-galactose, but it may also activate other hexose- or pentose-1-phosphates such as galacturonic acid-1-phosphate or arabinose-1-phosphate. In order to gain insight into structural features governing the differences in substrate specificity, we determined the crystal structure of the L. major USP in the APO-, UTP-, and UDP-sugar-bound conformations. The overall tripartite structure of USP exhibits a significant structural homology to other nucleotidyldiphosphate-glucose pyrophosphorylases. The obtained USP structures reveal the structural rearrangements occurring during the stepwise binding process of the substrates. Moreover, the different product complexes explain the broad substrate specificity of USP, which is enabled by structural changes in the sugar binding region of the active site.
Asunto(s)
Glucofosfatos/metabolismo , Leishmania major/enzimología , Fosfatos de Azúcar/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Secuencia de Aminoácidos , Catálisis , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis.
Asunto(s)
Leishmania major/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Leishmania major/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Eliminación de Secuencia , Transducción de Señal , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Galactosa/química , Uridina Difosfato Glucosa/químicaRESUMEN
The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.
Asunto(s)
Galactosafosfatos/metabolismo , Leishmania major/enzimología , Nucleotidiltransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Uridina Difosfato/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Molecular , Galactosafosfatos/genética , Glucofosfatos/genética , Glucofosfatos/metabolismo , Glicocálix/enzimología , Glicocálix/genética , Leishmania major/genética , Datos de Secuencia Molecular , Nucleotidiltransferasas/genética , Proteínas Protozoarias/genética , Especificidad por Sustrato/fisiología , Uridina Difosfato/genética , Uridina Trifosfato/genética , Uridina Trifosfato/metabolismoRESUMEN
The master circadian pacemaker of the suprachiasmatic nuclei coordinates behavioral and physiological rhythms via synchronization of subordinate peripheral oscillators in the central nervous system and organs throughout the body. Among these organs, the adrenal glands hold a prime position because of their regulatory influence on numerous physiological functions via rhythmic secretion of catecholamines and corticoid hormones into the bloodstream. In this report, the authors perform whole genome microarray hybridization to characterize the circadian transcriptome of the murine adrenal. They show that ~5% of the mouse genome is under circadian control in this gland. Using gene ontology analysis, they identify classes of transcripts that may synchronize adrenal hormone production. The authors' expression profiling also revealed that multiple histone genes implicated in either DNA replication or transcriptional regulation are clock controlled, suggesting a novel way by which the circadian clock may regulate the chromatin state.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Ritmo Circadiano , Perfilación de la Expresión Génica , Nucleosomas/metabolismo , Algoritmos , Animales , Conducta Animal , Cromatina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Oscilometría , Esteroides/química , Núcleo Supraquiasmático/metabolismoRESUMEN
In mammals, the master clock of the suprachiasmatic nuclei (SCN) and subordinate clocks found throughout the body coordinate circadian rhythms of behavior and physiology. We characterize the clock of the adrenal, an important endocrine gland that synchronizes physiological and metabolic rhythms. Clock gene expression was detected in the outer adrenal cortex prefiguring a role of the clock in regulating gluco- and mineral corticoid biogenesis. In Per2/Cry1 double mutant mice, which lack a circadian clock, hypothalamus/pituitary/adrenal axis regulation was defective. Organ culture and tissue transplantation suggest that the adrenal pacemaker gates glucocorticoid production in response to adrenocorticotropin (ACTH). In vivo the adrenal circadian clock can be entrained by light. Transcriptome profiling identified rhythmically expressed genes located at diverse nodes of steroid biogenesis that may mediate gating of the ACTH response by the adrenal clock.
Asunto(s)
Corticoesteroides/metabolismo , Corteza Suprarrenal/química , Corteza Suprarrenal/metabolismo , Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Corticoesteroides/análisis , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Criptocromos , Flavoproteínas/genética , Flavoproteínas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Cultivo de Órganos , Proteínas Circadianas Period , Transducción de Señal , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle that transfers reduction equivalents from the cytosol into the mitochondrion. Within the testis, immunohistological analysis localized human mGPDH to late spermatids and to the midpiece of spermatozoa. The expression of human mGPDH is regulated by two somatic promoters, and here, we describe a third testis-specific promoter of human mGPDH. The usage of this testis-specific promoter correlates with the expression of a shortened mGPDH transcript of approximately 2.4 kb in length, which is solely detectable from testicular RNA. Within the testis-specific promoter, we detected a cAMP-response element (CRE) site at -51, which binds the testis-specific transcriptional activator CRE modulator tau (CREMtau) in electrophoretic mobility shift assays. This recognition site overlaps with a nuclear receptor binding half-site at -49, which binds the testis-specific transcriptional repressor germ cell nuclear factor (GCNF). Both factors compete for binding to the same DNA response element. Ectopic expression of CREMtau in HepG2 cells activated a promoter-driven luciferase construct in transient transfection experiments. Additional cotransfection of GCNF relieved this activity, suggesting a down-regulation of CREMtau-mediated activation by GCNF. This effect was preserved by introducing the CRE/nuclear receptor-binding element into a heterologous promoter context. Our data suggest a down-regulation of CREMtau-mediated gene expression by GCNF, which might be a general regulation mechanism for several postmeiotically expressed genes with a temporal expression peak during early spermatid development.