RESUMO
The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/agonistas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Ativação Transcricional/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Linhagem Celular Tumoral , Cromatina/metabolismo , Feminino , Genoma , Humanos , Ligantes , Fígado/metabolismo , Receptores X do Fígado/química , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Elementos de Resposta/genéticaRESUMO
Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400-657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.
Assuntos
ADP Ribose Transferases/genética , Cisteína/genética , Mutação de Sentido Incorreto , Poli(ADP-Ribose) Polimerases/genética , ADP Ribose Transferases/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Animais , Biocatálise/efeitos dos fármacos , Células COS , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Chlorocebus aethiops , Cisteína/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Células MCF-7 , Proteínas de Transporte de Nucleosídeos , Poli(ADP-Ribose) Polimerases/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Dedos de Zinco/genéticaRESUMO
Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRß activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/ß peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRß co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRß. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1.
Assuntos
ADP Ribose Transferases/metabolismo , Núcleo Celular/metabolismo , Receptores Nucleares Órfãos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , ADP Ribose Transferases/genética , Adenosina Difosfato Ribose/genética , Adenosina Difosfato Ribose/metabolismo , Animais , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Células Hep G2 , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Receptores X do Fígado , Camundongos , Camundongos Knockout , Proteínas de Transporte de Nucleosídeos , Receptores Nucleares Órfãos/genética , Poli(ADP-Ribose) Polimerases/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.