Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol

The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl-CoA reductase produces mevalonate, which is converted to sterols and to other products, including geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and rapidly degraded when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Here, we use RNA interference to show that sterol-accelerated ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the endoplasmic reticulum that were shown previously to accelerate degradation of reductase when overexpressed by transfection. Alanine substitution experiments reveal that binding of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a sterol-stimulated fashion. When lysine 248 of reductase is substituted with arginine, Insig binding persists, but the reductase is no longer ubiquitinated and degradation is markedly slowed. Lysine 248 is predicted to lie immediately adjacent to a membrane-spanning helix, suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show that Insig-dependent, sterol-stimulated degradation of reductase is further accelerated when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-carbon farnesol, raising the possibility that the nonsterol potentiator of reductase regulation is a geranylgeranylated protein.     

Construct synthetic gene encoding artificial spider dragline silk protein and its expression in milk of transgenic mice

Based on the known partial cDNA sequence of dragline silk protein an artificial gene monomer, a 360 bp sequence, was designed and polymerized to encode an analog of dragline silk protein. Six tandem copies of monomer were cloned into pBC1 vector and microinjected into the pronuclei of fertilized Kunming White eggs. Transgenic mice were screened by Polymerase Chain Reaction (PCR) and Southern blot which revealed that 10 mice (5 male, 5 female) among 58 mice were transgenic positive. Milk of five F0 mice and eight F1 mice was analyzed by Western blot, and two F0 mice and seven F1 mice expressed recombinant dragline silk protein. In transgenic mice milk a maximum of concentration of recombinant dragline silk protein was 11.7 mg/L by radioimmunoassay.     

高脂血症大鼠四氢生物蝶呤治疗后动脉血管过氧化脂质水平及力学性质改变

目的：探讨长期四氢生物蝶呤（BH4）治疗对高脂血症（HL）大鼠血管脂质过氧化水平及血管力学性质的影响。方法：选用8周龄雄性Wistar大鼠54只,随机分为3组（n=18）：对照组、高脂饮食组（HE组）和高脂饮食并腹腔注射BH4组（HL＋BH4组）,于第8、16和24周龄时每组各杀死6只大鼠测定血脂和脂质过氧化终产物丙二醛（MDA）水平,主动脉血管测量零应力状态张开角、压力-直径关系。结果：至B地治疗后的第16和24周龄,血脂无明显变化,但MDA明显降低（P〈0．01）;HL＋BH4组和HL组比较胸主动脉张开角显著减小（P〈0．01）、压力-直径（P-D）关系曲线上移。结论 BH4可以减轻由于长期高脂血症导致的脂质过氧化,恢复血管弹性,降低血管的结构异常改变。     

Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.     

Niemann-Pick C1-Like 1 and cholesterol uptake

Niemann-Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

Niemann–Pick C1-Like 1 and cholesterol uptake

Niemann–Pick C1-Like 1 (NPC1L1) is a polytopic transmembrane protein responsible for dietary cholesterol and biliary cholesterol absorption. Consistent with its functions, NPC1L1 distributes on the brush border membrane of enterocytes and the canalicular membrane of hepatocytes in humans. As the molecular target of ezetimibe, a hypocholesterolemic drug, its physiological and pathological significance has been recognized and intensively studied for years. Recently, plenty of new findings reveal the molecular mechanism of NPC1L1's role in cholesterol uptake, which may provide new insights on our understanding of cholesterol absorption. In this review, we summarized recent progress in these studies and proposed a working model, hoping to provide new perspectives on the regulation of cholesterol transport and metabolism.     

Licochalcone A Attenuates Chronic Neuropathic Pain in Rats by Inhibiting Microglia Activation and Inflammation

Neurochemical Research - Immune response plays a vital role in the pathogenesis of neuropathic pain. Immune response-targeted therapy becomes an effective strategy for treating neuropathic pain....