RESUMO
SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response.
Assuntos
Hipoglicemiantes/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Polimorfismo de Nucleotídeo Único , Tecido Adiposo , Animais , Expressão Gênica , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismoRESUMO
Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPß, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPß's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPß from PARylation-mediated inhibition. This promotes the binding of C/EBPß at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPß, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes.
Assuntos
Adipócitos/enzimologia , Adipogenia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células-Tronco Embrionárias/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , DNA/genética , DNA/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células NIH 3T3 , Fenótipo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios Proteicos , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , TransfecçãoRESUMO
Mediator activates RNA polymerase II (Pol II) function during transcription, but it remains unclear whether Mediator is able to travel with Pol II and regulate Pol II transcription beyond the initiation and early elongation steps. By using in vitro and in vivo transcription recycling assays, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9 (CDK9), dynamically moves along with Pol II throughout the transcribed genes to drive Pol II recycling after the initial round of transcription. Mechanistically, MED31 mediates the recycling of phosphorylated MED1 and Pol II, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth by decreasing MED1 phosphorylation and Pol II recycling. Our results reveal a novel role of MED1 in Pol II transcription and identify phosphorylated MED1 as a targetable driver of dysregulated Pol II recycling in cancer.
Assuntos
Neoplasias , RNA Polimerase II , Animais , Humanos , Masculino , Mamíferos/genética , Complexo Mediador/metabolismo , Subunidade 1 do Complexo Mediador/genética , Neoplasias/genética , Fosforilação , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
RATIONALE: Single-nucleotide polymorphisms near the ILRUN (inflammation and lipid regulator with ubiquitin-associated-like and NBR1 [next to BRCA1 gene 1 protein]-like domains) gene are genome-wide significantly associated with plasma lipid traits and coronary artery disease (CAD), but the biological basis of this association is unknown. OBJECTIVE: To investigate the role of ILRUN in plasma lipid and lipoprotein metabolism. METHODS AND RESULTS: ILRUN encodes a protein that contains a ubiquitin-associated-like domain, suggesting that it may interact with ubiquitinylated proteins. We generated mice globally deficient for Ilrun and found they had significantly lower plasma cholesterol levels resulting from reduced liver lipoprotein production. Liver transcriptome analysis uncovered altered transcription of genes downstream of lipid-related transcription factors, particularly PPARα (peroxisome proliferator-activated receptor alpha), and livers from Ilrun-deficient mice had increased PPARα protein. Human ILRUN was shown to bind to ubiquitinylated proteins including PPARα, and the ubiquitin-associated-like domain of ILRUN was found to be required for its interaction with PPARα. CONCLUSIONS: These findings establish ILRUN as a novel regulator of lipid metabolism that promotes hepatic lipoprotein production. Our results also provide functional evidence that ILRUN may be the casual gene underlying the observed genetic associations with plasma lipids at 6p21 in human.
Assuntos
Hepatócitos/metabolismo , Lipoproteínas/sangue , Fígado/metabolismo , Animais , Glicemia/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , HDL-Colesterol/sangue , HDL-Colesterol/genética , Regulação da Expressão Gênica , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Células HEK293 , Humanos , Lipoproteínas/genética , Lipoproteínas VLDL/sangue , Lipoproteínas VLDL/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Ligação Proteica , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Transcriptoma , Triglicerídeos/sangue , Triglicerídeos/genética , UbiquitinaçãoRESUMO
PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene's transcription start site (TSS) in human but not mouse adipocytes. The -4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.
Assuntos
Adipócitos/metabolismo , Amidoidrolases/metabolismo , PPAR gama/metabolismo , Tecido Adiposo/metabolismo , Amidoidrolases/genética , Animais , Expressão Gênica , Regulação da Expressão Gênica , Variação Genética , Humanos , Masculino , Camundongos , Obesidade/genética , Fenótipo , TiazolidinedionasRESUMO
HER2, which is associated with clinically aggressive disease, is overexpressed in 15-20% of breast cancers (BC). The host immune system participates in the therapeutic response of HER2+ breast cancer. Identifying genetic programs that participate in ErbB2-induced tumors may provide the rational basis for co-extinction therapeutic approaches. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Herein, genetic deletion of endogenous Pparγ1 restrained mammary tumor progression, lipogenesis, and induced local mammary tumor macrophage infiltration, without affecting other tissue hematopoietic stem cell pools. Endogenous Pparγ1 induced expression of both an EphA2-Amphiregulin and an inflammatory INFγ and Cxcl5 signaling module, that was recapitulated in human breast cancer. Pparγ1 bound directly to growth promoting and proinflammatory target genes in the context of chromatin. We conclude Pparγ1 promotes ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction. Herein, endogenous Pparγ1 promoted ErbB2-mediated mammary tumor onset and progression. PPARγ1 increased expression of an EGF-EphA2 receptor tyrosine kinase module and a cytokine/chemokine 1 transcriptional module. The induction of a pro-tumorigenic inflammatory state by Pparγ1 may provide the rationale for complementary coextinction programs in ErbB2 tumors.
RESUMO
Brown adipose tissue (BAT) generates heat to maintain body temperature and suppress obesity. Agonists for nuclear receptors PPARα and PPARγ both affect brown adipocyte function, yet the interplay between these factors in BAT is uncertain. Here, we report that PPARα shares most genomic binding sites with PPARγ, and these common binding sites are more related to BAT function than PPARγ-selective sites without PPARα. Integrating PPARα and PPARγ genomic occupancy with cold-responsive BAT transcriptomes identifies a subset of 16 genes with potential relevance to BAT function. Among these, we focused on the lysosomal protease cathepsin Z (CTSZ) and showed it is necessary for mitochondrial respiration in both mouse and human brown adipocytes. Thus, CTSZ is a shared PPARα/γ target gene in BAT and a regulator of brown adipocyte thermogenic function.
Assuntos
Adipócitos Marrons/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , Termogênese/genética , Tecido Adiposo Marrom/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Catepsina Z/genética , Catepsina Z/metabolismo , Temperatura Baixa , Genoma , Humanos , Masculino , Camundongos Endogâmicos C57BL , PPAR alfa/agonistas , PPAR gama/agonistas , Ligação ProteicaRESUMO
Thiazolidinedione drugs (TZDs) target the transcriptional activity of peroxisome proliferator activated receptor γ (PPARγ) to reverse insulin resistance in type 2 diabetes, but side effects limit their clinical use. Here, using human adipose stem cell-derived adipocytes, we demonstrate that SNPs were enriched at sites of patient-specific PPARγ binding, which correlated with the individual-specific effects of the TZD rosiglitazone (rosi) on gene expression. Rosi induction of ABCA1, which regulates cholesterol metabolism, was dependent upon SNP rs4743771, which modulated PPARγ binding by influencing the genomic occupancy of its cooperating factor, NFIA. Conversion of rs4743771 from the inactive SNP allele to the active one by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated editing rescued PPARγ binding and rosi induction of ABCA1 expression. Moreover, rs4743771 is a major determinant of undesired serum cholesterol increases in rosi-treated diabetics. These data highlight human genetic variation that impacts PPARγ genomic occupancy and patient responses to antidiabetic drugs, with implications for developing personalized therapies for metabolic disorders.
Assuntos
Adipócitos/citologia , Tecido Adiposo/citologia , Variação Genética , Hipoglicemiantes/farmacologia , Células-Tronco/citologia , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adulto , Idoso , Sequência de Bases , Linhagem Celular , Colesterol/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Edição de Genes , Loci Gênicos , Humanos , Hipoglicemiantes/uso terapêutico , Pessoa de Meia-Idade , Fatores de Transcrição NFI/metabolismo , PPAR gama/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica/efeitos dos fármacos , Rosiglitazona/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Long intergenic noncoding RNAs (lincRNAs) have emerged as important modulators of cellular functions. Most lincRNAs are not conserved among mammals, raising the fundamental question of whether nonconserved adipose-expressed lincRNAs are functional. To address this, we performed deep RNA sequencing of gluteal subcutaneous adipose tissue from 25 healthy humans. We identified 1001 putative lincRNAs expressed in all samples through de novo reconstruction of noncoding transcriptomes and integration with existing lincRNA annotations. One hundred twenty lincRNAs had adipose-enriched expression, and 54 of these exhibited peroxisome proliferator-activated receptor γ (PPARγ) or CCAAT/enhancer binding protein α (C/EBPα) binding at their loci. Most of these adipose-enriched lincRNAs (~85%) were not conserved in mice, yet on average, they showed degrees of expression and binding of PPARγ and C/EBPα similar to those displayed by conserved lincRNAs. Most adipose lincRNAs differentially expressed (n = 53) in patients after bariatric surgery were nonconserved. The most abundant adipose-enriched lincRNA in our subcutaneous adipose data set, linc-ADAL, was nonconserved, up-regulated in adipose depots of obese individuals, and markedly induced during in vitro human adipocyte differentiation. We demonstrated that linc-ADAL interacts with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) at distinct subcellular locations to regulate adipocyte differentiation and lipogenesis.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , RNA Longo não Codificante/metabolismo , Adipócitos/citologia , Diferenciação Celular , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Lipídeos/biossíntese , Lipogênese , PPAR gama/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição GênicaRESUMO
Obesity causes insulin resistance, and PPARγ ligands such as rosiglitazone are insulin sensitizing, yet the mechanisms remain unclear. In C57BL/6 (B6) mice, obesity induced by a high-fat diet (HFD) has major effects on visceral epididymal adipose tissue (eWAT). Here, we report that HFD-induced obesity in B6 mice also altered the activity of gene regulatory elements and genome-wide occupancy of PPARγ. Rosiglitazone treatment restored insulin sensitivity in obese B6 mice, yet, surprisingly, had little effect on gene expression in eWAT. However, in subcutaneous inguinal fat (iWAT), rosiglitazone markedly induced molecular signatures of brown fat, including the key thermogenic gene Ucp1. Obesity-resistant 129S1/SvImJ mice (129 mice) displayed iWAT browning, even in the absence of rosiglitazone. The 129 Ucp1 locus had increased PPARγ binding and gene expression that were preserved in the iWAT of B6x129 F1-intercrossed mice, with an imbalance favoring the 129-derived alleles, demonstrating a cis-acting genetic difference. Thus, B6 mice have genetically defective Ucp1 expression in iWAT. However, when Ucp1 was activated by rosiglitazone, or by iWAT browning in cold-exposed or young mice, expression of the B6 version of Ucp1 was no longer defective relative to the 129 version, indicating epigenomic rescue. These results provide a framework for understanding how environmental influences like drugs can affect the epigenome and potentially rescue genetically determined disease phenotypes.
Assuntos
Epigênese Genética , Obesidade/metabolismo , PPAR gama/fisiologia , Animais , Dieta Hiperlipídica/efeitos adversos , Hipoglicemiantes/farmacologia , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Ligação Proteica , Elementos Reguladores de Transcrição , Rosiglitazona , Gordura Subcutânea Abdominal/metabolismo , Tiazolidinedionas/farmacologia , Ativação Transcricional , Transcriptoma , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Intracellular cholesterol transport is essential for the maintenance of cholesterol homeostasis. Many aspects of cholesterol metabolism are well-known, including its synthesis in the endoplasmic reticulum, its extracellular transport in plasma lipoproteins, its uptake by the low-density lipoprotein receptor, and its regulation of SREBP and LXR transcription factors. These fundamental pathways in cholesterol metabolism all rely on its proper intracellular distribution among subcellular organelles and the plasma membrane. Transport involving the ER and endosomes is essential for cholesterol synthesis, uptake, and esterification, whereas cholesterol catabolism by enzymes in mitochondria and ER generates steroids, bile acids, and oxysterols. Cholesterol is a highly hydrophobic lipid that requires specialized transport in the aqueous cytosol, involving either vesicles or nonvesicular mechanisms. The latter includes hydrophobic cavity transporters such as StAR-related lipid transfer (START) proteins. Molecular understanding of intracellular cholesterol trafficking has lagged somewhat behind other aspects of cholesterol metabolism, but recent advances have defined some transport pathways and candidate proteins. In this review, we discuss cholesterol transport among specific intracellular compartments, emphasizing the relevance of these pathways to cholesterol homeostasis.
Assuntos
Colesterol/metabolismo , Líquido Intracelular/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Homeostase , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Circadian and metabolic physiology are intricately intertwined, as illustrated by Rev-erbα, a transcription factor (TF) that functions both as a core repressive component of the cell-autonomous clock and as a regulator of metabolic genes. Here, we show that Rev-erbα modulates the clock and metabolism by different genomic mechanisms. Clock control requires Rev-erbα to bind directly to the genome at its cognate sites, where it competes with activating ROR TFs. By contrast, Rev-erbα regulates metabolic genes primarily by recruiting the HDAC3 co-repressor to sites to which it is tethered by cell type-specific transcription factors. Thus, direct competition between Rev-erbα and ROR TFs provides a universal mechanism for self-sustained control of the molecular clock across all tissues, whereas Rev-erbα uses lineage-determining factors to convey a tissue-specific epigenomic rhythm that regulates metabolism tailored to the specific need of that tissue.
Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Metabolismo/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Animais , Fator 6 Nuclear de Hepatócito/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Especificidade de Órgãos , Ligação Proteica , Distribuição TecidualRESUMO
Type 2 diabetes is caused by insulin resistance coupled with an inability to produce enough insulin to control blood glucose, and thiazolidinediones (TZDs) are the only current antidiabetic agents that function primarily by increasing insulin sensitivity. However, despite clear benefits in glycemic control, this class of drugs has recently fallen into disuse due to concerns over side effects and adverse events. Here we review the clinical data and attempt to balance the benefits and risks of TZD therapy. We also examine potential mechanisms of action for the beneficial and harmful effects of TZDs, mainly via agonism of the nuclear receptor PPARγ. Based on critical appraisal of both preclinical and clinical studies, we discuss the prospect of harnessing the insulin sensitizing effects of PPARγ for more effective, safe, and potentially personalized treatments of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/metabolismo , Tiazolidinedionas/uso terapêutico , Animais , Doenças Cardiovasculares/tratamento farmacológico , Edema/etiologia , Fraturas Ósseas/tratamento farmacológico , Humanos , Hipoglicemiantes/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , PPAR gama/agonistas , PPAR gama/metabolismo , Tiazolidinedionas/efeitos adversos , Neoplasias da Bexiga Urinária/tratamento farmacológicoRESUMO
The winged helix protein FOXA2 and the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) are highly conserved, regionally expressed transcription factors (TFs) that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for Foxa2 in mouse liver and PPARγ in mouse adipocytes has previously produced consensus-binding sites that are nearly identical to those used by the corresponding TFs in human cells. We report here that, despite the conservation of the canonical binding motif, the great majority of binding regions for FOXA2 in human liver and for PPARγ in human adipocytes are not in the orthologous locations corresponding to the mouse genome, and vice versa. Of note, TF binding can be absent in one species despite sequence conservation, including motifs that do support binding in the other species, demonstrating a major limitation of in silico binding site prediction. Whereas only approximately 10% of binding sites are conserved, gene-centric analysis reveals that about 50% of genes with nearby TF occupancy are shared across species for both hepatic FOXA2 and adipocyte PPARγ. Remarkably, for both TFs, many of the shared genes function in tissue-specific metabolic pathways, whereas species-unique genes fail to show enrichment for these pathways. Nonetheless, the species-unique genes, like the shared genes, showed the expected transcriptional regulation by the TFs in loss-of-function experiments. Thus, species-specific strategies underlie the biological functions of metabolic TFs that are highly conserved across mammalian species. Analysis of factor binding in multiple species may be necessary to distinguish apparent species-unique noise and reveal functionally relevant information.
Assuntos
Sequência Conservada/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , PPAR gama/metabolismo , Animais , Sequência de Bases/genética , Sítios de Ligação/genética , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/química , Fator 3-beta Nuclear de Hepatócito/genética , Humanos , Camundongos , Análise em Microsséries , PPAR gama/química , PPAR gama/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
CONTEXT: Nonclassic congenital lipoid adrenal hyperplasia (lipoid CAH) is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function. Affected individuals can present with a phenotype of late onset adrenal insufficiency with only mild or minimally disordered sexual development. OBJECTIVES: The aim was to delineate the clinical spectrum of StAR mutations and correlate phenotype with StAR activity. PATIENTS: Four patients had nonclassic/atypical lipoid CAH. Adrenal insufficiency was manifested at birth in two patients and at 11 months and 4 yr in the other two. Three were 46,XY with underdeveloped genitalia. METHODS: The StAR gene was sequenced, mutations were recreated in expression vectors, and StAR activity was measured as pregnenolone production in COS-1 cells cotransfected with the cholesterol side-chain cleavage system. StAR mutants were expressed as N-62 StAR in bacteria, and purified proteins were tested for activity with isolated steroidogenic mitochondria and for cholesterol-binding capacity. RESULTS: DNA sequencing identified mutations on all alleles. Missense mutations were R188C, G221D, L260P, and F267S; we also tested R192C described by others. The respective activities of R188C, R192C, G221D, L260P, and F267S were 8.0, 39.4, 2.4, 3.1, and 6.1% of wild-type in transfected cells, and 12.8, 54.8, 6.3, 1.8, and 9.5% with isolated mitochondria. Cholesterol binding capacities of R188C, R192C, G221D, L260P, and F267S were 6.7, 55.3, 10.2, 4.6, and 20.9%. These data are correlated to the three-dimensional structure of StAR. CONCLUSIONS: There is a broad clinical spectrum of StAR mutations; StAR activities in vitro correlate well with clinical phenotypes.
Assuntos
Hiperplasia Suprarrenal Congênita/genética , Fenótipo , Fosfoproteínas/genética , Adolescente , Hiperplasia Suprarrenal Congênita/metabolismo , Insuficiência Adrenal/genética , Insuficiência Adrenal/metabolismo , Adulto , Pré-Escolar , Colesterol/genética , Colesterol/metabolismo , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação/genética , Fosfoproteínas/metabolismoRESUMO
The StarD4 and StarD5 proteins share approximately 30% identity, and each is a steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. We previously showed StarD4 expression is sterol-repressed, consistent with regulation by sterol regulatory element-binding proteins (SREBPs), whereas StarD5 is not sterol-regulated. Here we further address the regulation and function of StarD4 and StarD5. Unlike StAR, the START family prototype, StarD4 and StarD5 were not induced by steroidogenic stimuli in Leydig cells. However, StarD4 and StarD5 showed StAR-like activity in a cell culture steroidogenesis assay, indicating cholesterol transfer. In transgenic mice expressing active SREBPs, StarD4 was predominantly activated by SREBP-2 rather than SREBP-1a. The mouse and human StarD4 proximal promoters share approximately 70% identity, including several potential sterol regulatory elements (SREs). Reporters driven by the StarD4 promoter from either species were transfected into NIH-3T3 cells, and reporter activity was highly repressed by sterols. Site-directed mutagenesis of potential SREs identified a conserved functional SRE in the mouse (TCGGTCCAT) and human (TCATTCCAT) promoters. StarD5 was not sterol-repressed via SREBPs nor was it sterol-activated via liver X receptors (LXRs). Even though StarD4 and StarD5 were not LXR targets, their overexpression stimulated LXR reporter activity, suggesting roles in cholesterol metabolism. StarD5 expression increased 3-fold in free cholesterol-loaded macrophages, which activate the endoplasmic reticulum (ER) stress response. When NIH-3T3 cells were treated with agents to induce ER stress, StarD5 expression increased 6-8-fold. Because StarD4 is regulated by sterols via SREBP-2, whereas StarD5 is activated by ER stress, they likely serve distinct functions in cholesterol metabolism.
Assuntos
Colesterol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células COS , Proteínas de Transporte , Clonagem Molecular , DNA Complementar/metabolismo , Ensaio de Imunoadsorção Enzimática , Genes Reporter , Humanos , Fígado/metabolismo , Receptores X do Fígado , Luciferases/metabolismo , Macrófagos/metabolismo , Masculino , Proteínas de Membrana Transportadoras/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Receptores Nucleares Órfãos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Esteróis/metabolismo , Transfecção , TransgenesRESUMO
The x-ray structure of the mouse cholesterol-regulated START protein 4 (StarD4) has been determined at 2.2-A resolution, revealing a compact alpha/beta structure related to the START domain present in the cytoplasmic C-terminal portion of human MLN64. The volume of the putative lipid-binding tunnel was estimated at 847 A(3), which is consistent with the binding of one cholesterol-size lipid molecule. Comparison of the tunnel-lining residues in StarD4 and MLN64-START permitted identification of possible lipid specificity determinants in both molecular tunnels. Homology modeling of related proteins, and comparison of the StarD4 and MLN64-START structures, showed that StarD4 is a member of a large START domain superfamily characterized by the helix-grip fold. Additional mechanistic and evolutionary studies should be facilitated by the availability of a second START domain structure from a distant relative of MLN64.
Assuntos
Proteínas de Transporte/química , Colesterol/metabolismo , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Animais , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Simulação por Computador , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosfoproteínas , Filogenia , Estrutura Terciária de Proteína , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
High-cholesterol diets elicit changes in gene expression via such transcription factors as sterol-regulatory element binding proteins (SREBPs) and liver X receptors (LXRs). We used Affymetrix microarrays to identify genes in mouse liver regulated by dietary cholesterol (0.0% vs. 0.5% cholesterol wt/wt). Three independent experiments were performed, and data were analyzed with Affymetrix Microarray Suite and ANOVA statistical software. There were 69 unique Unigene clusters consistently regulated by dietary cholesterol (37 downregulated and 32 upregulated). The array results were confirmed by quantitative RT-PCR (Q-PCR) for seven of nine downregulated genes and five of six upregulated genes. A time course of dietary cholesterol feeding over 1 week revealed different temporal patterns of gene regulation for these confirmed genes. Six downregulated genes were examined in transgenic mice overexpressing truncated nuclear forms of SREBP-1a and SREBP-2, and all were induced in these mice. A second microarray analysis of mice treated with the LXR agonist TO901317 confirmed that 13 of the 32 cholesterol upregulated genes were also LXR-activated. This array result was confirmed by Q-PCR for three of three genes. In summary, these studies identified and confirmed six novel dietary cholesterol-regulated genes, three putative SREBP target genes (calcium/calmodulin-dependent protein kinase 1D, fatty acid binding protein 5, and proprotein convertase subtilisin/kexin 9), and three putative LXR target genes (a disintegrin and metalloprotease domain 11, apoptosis-inhibitory 6, and F-box-only protein 3).
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Colesterol na Dieta/farmacologia , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Colesterol na Dieta/administração & dosagem , Regulação para Baixo/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres Sexuais , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2RESUMO
Using cDNA microarrays, we identified StarD4 as a gene whose expression decreased more than 2-fold in the livers of mice fed a high-cholesterol diet. StarD4 expression in cultured 3T3 cells was also sterol-regulated, and known sterol regulatory element binding protein (SREBP)-target genes showed coordinate regulation. The closest homologues to StarD4 were two other StAR-related lipid transfer (START) proteins named StarD5 and StarD6. StarD4, StarD5, and StarD6 are 205- to 233-aa proteins consisting almost entirely of START domains. These three constitute a subfamily among START proteins, sharing approximately 30% amino acid identity with one another, approximately 20% identity with the cholesterol-binding START domains of StAR and MLN64, and less than 15% identity with phosphatidylcholine transfer protein (PCTP) and other START domains. StarD4 and StarD5 were expressed in most tissues, with highest levels in liver and kidney, whereas StarD6 was expressed exclusively in the testis. In contrast to StarD4, expression of StarD5 and MLN64 was not sterol-regulated. StarD4, StarD5, and StarD6 may be involved in the intracellular transport of sterols or other lipids.