RESUMO
Hedgehog (Hh) signaling pathway plays a pivotal role in embryonic development. Hh binding to Patched1 (PTCH1) derepresses Smoothened (SMO), thereby activating the downstream signal transduction. Covalent SMO modification by cholesterol in its cysteine-rich domain (CRD) is essential for SMO function. SMO cholesterylation is a calcium-accelerated autoprocessing reaction, and STIM1-ORAI1-mediated store-operated calcium entry promotes cholesterylation and activation of endosome-localized SMO. However, it is unknown whether the Hh-PTCH1 interplay regulates the activity of the endoplasmic reticulum (ER)-localized SMO. Here, we found that PTCH1 inhibited the COPII-dependent export of SMO from the ER, whereas Hh promoted this process. The RRxWxR amino acid motif in the cytosolic tail of SMO was essential for COPII recognition, ciliary localization, and signal transduction activity. Hh and PTCH1 regulated cholesterol modification of the ER-localized SMO, and SMO cholesterylation accelerated its exit from ER. The GRAMD1/ASTER sterol transport proteins facilitated cholesterol transfer to ER from PM, resulting in increased SMO cholesterylation and enhanced Hh signaling. Collectively, we reveal a regulatory role of GRAMD-mediated cholesterol transport in ER-resident SMO maturation and Hh signaling.
Assuntos
Cálcio , Proteínas Hedgehog , Transporte Biológico , Cálcio/metabolismo , Colesterol/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Membrana/metabolismoRESUMO
High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.
Assuntos
Receptor de Asialoglicoproteína , Colesterol , Metabolismo dos Lipídeos , Proteínas Quinases Ativadas por AMP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Receptor de Asialoglicoproteína/antagonistas & inibidores , Receptor de Asialoglicoproteína/deficiência , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas/metabolismo , Atorvastatina/farmacologia , Proteína BRCA1 , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Colesterol/metabolismo , Sinergismo Farmacológico , Endocitose , Ezetimiba/farmacologia , Humanos , Lipídeos/análise , Lipídeos/sangue , Fígado/metabolismo , Receptores X do Fígado/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: High blood cholesterol accelerates the progression of atherosclerosis, which is an asymptomatic process lasting for decades. Rupture of atherosclerotic plaques induces thrombosis, which results in myocardial infarction or stroke. Lowering cholesterol levels is beneficial for preventing atherosclerotic cardiovascular disease. METHODS: Low-density lipoprotein (LDL) receptor (LDLR) was used as bait to identify its binding proteins in the plasma, and the coagulation factor prekallikrein (PK; encoded by the KLKB1 gene) was revealed. The correlation between serum PK protein content and lipid levels in young Chinese Han people was then analyzed. To investigate the effects of PK ablation on LDLR and lipid levels in vivo, we genetically deleted Klkb1 in hamsters and heterozygous Ldlr knockout mice and knocked down Klkb1 using adeno-associated virus-mediated shRNA in rats. The additive effect of PK and proprotein convertase subtilisin/kexin 9 inhibition also was evaluated. In addition, we applied the anti-PK neutralizing antibody that blocked the PK and LDLR interaction in mice. Mice lacking both PK and apolipoprotein e (Klkb1-/-Apoe-/-) were generated to assess the role of PK in atherosclerosis. RESULTS: PK directly bound LDLR and induced its lysosomal degradation. The serum PK concentrations positively correlated with LDL cholesterol levels in 198 young Chinese Han adults. Genetic depletion of Klkb1 increased hepatic LDLR and decreased circulating cholesterol in multiple rodent models. Inhibition of proprotein convertase subtilisin/kexin 9 with evolocumab further decreased plasma LDL cholesterol levels in Klkb1-deficient hamsters. The anti-PK neutralizing antibody could similarly lower plasma lipids through upregulating hepatic LDLR. Ablation of Klkb1 slowed the progression of atherosclerosis in mice on Apoe-deficient background. CONCLUSIONS: PK regulates circulating cholesterol levels through binding to LDLR and inducing its lysosomal degradation. Ablation of PK stabilizes LDLR, decreases LDL cholesterol, and prevents atherosclerotic plaque development. This study suggests that PK is a promising therapeutic target to treat atherosclerotic cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , LDL-Colesterol/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/prevenção & controle , Pré-Calicreína/deficiência , Receptores de LDL/metabolismo , Animais , Aterosclerose/genética , LDL-Colesterol/genética , Lisossomos/genética , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genética , Pré-Calicreína/metabolismo , Proteólise , Receptores de LDL/genéticaRESUMO
Insig-2 is an ER membrane protein negatively controlling lipid biosynthesis. Here, we find that Insig-2 is increased in the tissues, including liver, but unaltered in the muscle of gp78-deficient mice. In hepatocytes and undifferentiated C2C12 myoblasts, Insig-2 is ubiquitylated on Cys215 by gp78 and degraded. However, the C215 residue is oxidized by elevated reactive oxygen species (ROS) during C2C12 myoblasts differentiating into myotubes, preventing Insig-2 from ubiquitylation and degradation. The stabilized Insig-2 downregulates lipogenesis through inhibiting the SREBP pathway, helping to channel the carbon flux to ATP generation and protecting myotubes from lipid over-accumulation. Evolutionary analysis shows that the YECK (in which C represents Cys215 in human Insig-2) tetrapeptide sequence in Insig-2 is highly conserved in amniotes but not in aquatic amphibians and fishes, suggesting it may have been shaped by differential selection. Together, this study suggests that competitive oxidation-ubiquitylation on Cys215 of Insig-2 senses ROS and prevents muscle cells from lipid accumulation.
Assuntos
Cisteína/metabolismo , Proteínas de Membrana/metabolismo , Receptores do Fator Autócrino de Motilidade/metabolismo , Ubiquitinação , Anfíbios , Animais , Células CHO , Linhagem Celular , Cricetulus , Regulação para Baixo , Evolução Molecular , Peixes , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipogênese , Fígado/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Receptores do Fator Autócrino de Motilidade/genética , Análise de Sequência de Proteína , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , TranscriptomaRESUMO
Metabolism in mammals is regulated by complex interplay among different organs. Fatty acid synthesis is increased in white adipose tissue (WAT) when it is inhibited in the liver. Here we identify glycoprotein non-metastatic melanoma protein B (Gpnmb) as one liver-WAT cross-talk factor involved in lipogenesis. Inhibition of the hepatic sterol regulatory element-binding protein pathway leads to increased transcription of Gpnmb and promotes processing of the membrane protein to a secreted form. Gpnmb stimulates lipogenesis in WAT and exacerbates diet-induced obesity and insulin resistance. In humans, Gpnmb is tightly associated with body mass index and is a strong risk factor for obesity. Gpnmb inhibition by a neutralizing antibody or liver-specific knockdown improves metabolic parameters, including weight gain reduction and increased insulin sensitivity, probably by promoting the beiging of WAT. These results suggest that Gpnmb is a liver-secreted factor regulating lipogenesis in WAT, and that Gpnmb inhibition may provide a therapeutic strategy in obesity and diabetes.
Assuntos
Tecido Adiposo Branco/metabolismo , Proteínas do Olho/metabolismo , Resistência à Insulina , Fígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Obesidade/metabolismo , Animais , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Lipogênese/genética , Lipogênese/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores do Fator Autócrino de Motilidade/genética , Receptores do Fator Autócrino de Motilidade/metabolismo , Regulação para CimaRESUMO
Statins are inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, and have been clinically used to treat cardiovascular disease. However, a paradoxical increase of reductase protein following statin treatment may attenuate the effect and increase the side effects. Here we present a previously unexplored strategy to alleviate statin-induced reductase accumulation by inducing its degradation. Inspired by the observations that cholesterol intermediates trigger reductase degradation, we identify a potent degrader, namely Cmpd 81, through structure-activity relationship analysis of sterol analogs. Cmpd 81 stimulates ubiquitination and degradation of reductase in an Insig-dependent manner, thus dramatically reducing protein accumulation induced by various statins. Cmpd 81 can act alone or synergistically with statin to lower cholesterol and reduce atherosclerotic plaques in mice. Collectively, our work suggests that inducing reductase degradation by Cmpd 81 or similar chemicals alone or in combination with statin therapy can be a promising strategy for treating cardiovascular disease.
Assuntos
Colesterol/biossíntese , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Esteróis/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Sinergismo Farmacológico , Humanos , Masculino , Camundongos , Estrutura Molecular , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/prevenção & controle , Proteólise/efeitos dos fármacos , Esteróis/química , Ubiquitinação/efeitos dos fármacosRESUMO
Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.