葡萄糖感应器及其AMPK活性和细胞代谢状态的调控机理

葡萄糖是细胞的主要能量来源,它通过糖酵解和/或氧化代谢产生ATP。葡萄糖水平下降将激活AMP活化蛋白激酶(AMP-activated protein kinase-AMPK),数十年来,人们一直认为这是由于AMP或ADP的上升,或者说能量水平的下降引起的。厦门大学生命科学学院林圣彩研究团队与英国Dundee大学D.Grahame Hardie团队合作报道了一种通过感知胞内葡萄糖代谢中间物果糖-1,6-二磷酸(FBP)的下降来触发AMPK活化的机制:FBP水平下降,便不再占据其催化酶——醛缩酶(aldolase)上的催化位点,后者直接改变了溶酶体膜上的质子泵v-ATPase和与其相结合的\"Ragulator\"的构象,进而让携带有能磷酸化并激活AMPK的上游激酶LKB1的AXIN蛋白质转移到溶酶体膜表面,在此形成了能激活AMPK的复合体(该复合体也是由林圣彩实验室发现并鉴定-Cell Metabolism,2013,2014),从而激活AMPK,同时抑制了促进细胞进行生物合成和生长的激酶-mTORC1。重要的是,他们从中也颠覆性地发现细胞能量水平在葡萄糖水平急速下降期间仍保持不变,且AMPK上的AMP结合位点对于此时AMPK的活化不是必需的。该研究不仅发现了醛缩酶这一糖酵解酶的新功能:调控AMPK的葡萄糖感受器,还深刻地揭示了葡萄糖的本质:既是能量和物质来源,也是直接调控细胞生物合成和生长状态的信号,对多种代谢型疾病的发生、癌症与代谢关系的认知与治疗、卡路里限制与健康长寿等的认知具有重大意义。相关研究论文在2017年8月3日发表于《自然》[NATURE,548(7665):112-116,August 2017]

Revealing a steroid receptor ligand as a unique PPARγ agonist

过氧化物酶体增殖体激活受体γ（PPARγ）是核受体家族成员之一，它能够调节机体新陈代谢平衡，是国际上研发治疗糖尿病等代谢类疾病及抗癌药物的热门药物分子靶标。然而，许多副作用局限了这些配体药物的临床使用，如噻唑烷类（TZDs）PPARγ配体药物会导致体重增加、水肿、心力衰竭与肝中毒等。本课题通过研究前期筛选到的化合物米非司酮（RU-486），发现该化合物是一种新型PPARγ特异性类固醇激动剂。通过对PPARγ/RU-486复合体进行结晶、X射线衍射、对收集的衍射数据进行解析，我们从分子水平上揭示了这一独特的RU-486在PPARγ配体结合域内的特殊结合模型，从原子水平上解释了RU-486如何通过...Peroxisome proliferator activated receptor gamma (PPARγ) is a member of nuclear receptor family, which regulates metabolic homeostasis and is a hot molecular target for anti-diabetic and anti-cancer drugs research of the world. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPARγ agonist by High through put drug screening. We constructed the crystal of PPA...学位：理学硕士院系专业：生命科学学院生物医学科学系_生物化学与分子生物学学号：2172008115264

AMPK:不仅能感应细胞的能量状态还能感应葡萄糖

文章简介腺嘌呤核苷酸水平的改变,即AMP/ATP和ADP/ATP比值的上升是细胞能量状态下降的信号。众所周知,哺乳动物的AMPK会随细胞能量水平的下降而激活。这篇综述回顾了近来关于细胞的低能量状态如何激活AMPK的研究成果,同时还探讨了最近关于国家重点研发计划;;\n国家自然科学基金委项目的支

PITUITARY ONTOGENY OF THE SNELL DWARF MOUSE REVEALS PIT-1-INDEPENDENT AND PIT-1-DEPENDENT ORIGINS OF THE THYROTROPE

The anterior pituitary provides a model to study the molecular mechanisms responsible for emergence of distinct cell types within an organ. Dwarf mice (Snell) that express a mutant form of the tissue-specific POU-domain transcription factor Pit-1 fail to generate three cell types, including the thyrotrope (S. Li, E. B. Crenshaw, E. J. Rawson, D. S. Simmons, L. Swanson and M. G. Rosenfeld (1990), Nature 347, 528-533). Analyses of wild-type and Pit-1-defective mice, presented here, have revealed that thyrotropes unexpectedly arise from two independent cell populations. The first population is Pit-1-independent and appears on e12 in the rostral tip of the developing gland, but phenotypically disappears by the day of birth. The second is Pit-1-dependent and arises subsequently in the caudomedial portion of the developing gland (e15.5), following the initial expression of Pit-1 in this region. The failure of caudomedial thyrotrope cells to appear in the Snell dwarf, and the observation that Pit-1 can bind to and transactivate the TSH beta promoter, apparently enhanced by its phosphorylation, suggests that Pit-1 is directly required for the appearance of this distinct population that serves as the precursors of the mature thyrotrope cell type. These data suggest that different molecular mechanisms, based on the actions of distinct transcription factors, can serve to independently generate a specific cell phenotype during mammalian organogenesis

CDK5 activator p35 downregulates E-cadherin precursor independently of CDK5

Dysfunction of E-cadherins often results in metastasis of cancerous cells. Here we show that p35, a critical regulator of cyclin-dependent kinase 5 (CDK5), specifically depletes the precursor form of E-cadherin, but not the mature form, by using a precursor-specific antibody. Most intriguingly, this downregulation of precursor E-cadherin by p35 is unequivocally independent of CDK5. Moreover, we found that p35 forms complexes with E-cadherin proteins. We also found that p35 co-expression can target E-cadherin to lysosomes and that p35-triggered disappearance of E-cadherin precursor can be blocked specifically by lysosomal protease inhibitors, indicating that p35 induces endocytosis and subsequent degradation of precursor E-cadherin

Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or 2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux

SIRT7基因低表达胶质瘤细胞株代谢特征的NMR分析

肿瘤是一种代谢疾病,癌基因表达对肿瘤细胞代谢的影响是目前肿瘤研究的热点之一.本文利用基于核磁共振氢谱(~1H NMR)的代谢组学方法对癌基因SIRT7低表达胶质瘤细胞株的代谢特征进行分析,寻找与SIRT7基因表达相关的特征性代谢物和代谢通路.分析结果表明,SIRT7基因低表达组与对照组细胞的代谢轮廓存在显著性差异,其细胞水溶性萃取物中有22种代谢物浓度发生明显变化.与对照组相比,SIRT7基因低表达胶质瘤细胞株中乳酸、甘氨酸、谷氨酸等12种代谢物浓度升高;缬氨酸、亮氨酸、赖氨酸等10种代谢物浓度降低.通路富集分析提示氨酰-tRNA生物合成、氨基酸代谢等代谢通路与SIRT7低表达密切相关.以上结果为进一步阐明癌基因SIRT7调控胶质瘤细胞代谢的作用机制提供了理论依据.厦门市科技计划社会发展项目(3502Z20184064

Role of axin in nerve growth factor-stimulated neurite outgrowth

The role of integrin-linked kinase (ILK), a kinase that is involved in various cellular processes, including adhesion and migration, has not been studied in primary neurons. Using mRNA dot blot and Western blot analysis of ILK in rat and human brain tissue, we found that ILK is expressed in various regions of the CNS. Immunohistochemical and immunocytochemical techniques revealed granular ILK staining that is enriched in neurons and colocalizes with the 1 integrin subunit. The role of ILK in neurite growth promotion by NGF was studied in rat pheochromocytoma cells and dorsal root ganglion neurons using a pharmacological inhibitor of ILK (KP-392) or after overexpres-sion of dominant-negative ILK (ILK-DN). Both molecular and pharmacological inhibition of ILK activity significantly reduced NGF-induced neurite outgrowth. Survival assays indicate that KP-392-induced suppression of neurite outgrowth occurred in the absence of cell death. ILK kinase activity was stimulated by NGF. NGF-mediated stimulation of phosphorylation of both AKT and the Taukinase glycogen synthase kinase-3 (GSK-3) was inhibited in the presence of KP-392 and after overexpression of ILK-DN. Consequently, ILKinhibition resulted in an increase in the hyperphosphorylation of Tau , a substrate of GSK-3. Together these findings indicate that ILK is an important effector in NGF-mediated neurite outgrowth. The role of integrin-linked kinase (ILK), a kinase that is involved in various cellular processes, including adhesion and migration, has not been studied in primary neurons. Using mRNA dot blot and Western blot analysis of ILK in rat and human brain tissue, we found that ILK is expressed in various regions of the CNS. Immunohistochemical and immunocytochemical techniques revealed granular ILK staining that is enriched in neurons and colocalizes with the 1 integrin subunit. The role of ILK in neurite growth promotion by NGF was studied in rat pheochromocytoma cells and dorsal root ganglion neurons using a pharmacological inhibitor of ILK (KP-392) or after overexpres- sion of dominant-negative ILK (ILK-DN). Both molecular and pharmacological inhibition of ILK activity significantly reduced NGF- induced neurite outgrowth. Survival assays indicate that KP-392-induced suppression of neurite outgrowth occurred in the absence of cell death. ILK kinase activity was stimulated by NGF. NGF-mediated stimulation of phosphorylation of both AKT and the Tau kinase glycogen synthase kinase-3 (GSK-3) was inhibited in the presence of KP-392 and after overexpression of ILK-DN. Consequently, ILK inhibition resulted in an increase in the hyperphosphorylation of Tau , a substrate of GSK-3. Together these findings indicate that ILK is an important effector in NGF-mediated neurite outgrowth

Protein encoded by the Axin(Fu) allele effectively down-regulates wnt signaling but exerts a dominant negative effect on c-jun n-terminal kinase signaling

Axin plays an architectural role in many important signaling pathways that control various aspects of development and tumorigenesis, including the Wnt, transforming growth factor-beta, MAP kinase pathways, as well as p53 activation cascades. It is encoded by the mouse Fused (Fu) locus; the Axin(Fu) allele is caused by insertion of an IAP transposon. Axin(Fu/Fu) mice display varying phenotypes ranging from embryonic lethality to relatively normal adulthood with kinky tails. However, the protein product(s) has not been identified or characterized. In the present study, we conducted immunoprecipitation using brain extracts from the Axin(Fu) mice with specific antibodies against different regions of Axin and found that a truncated Axin containing amino acids 1-596 (designated as Axin(Fu-NT)) and the full-length complement of Axin (Axin(WT)) can both be generated from the AxinFu allele. When tested for functionality changes, Axin(Fu-NT) was found to abolish Axin-mediated activation of JNK, which plays a critical role in dorsoventral patterning. Together with a proteomics approach, we found that Axin(Fu-NT) contains a previously uncharacterized dimerization domain and can form a heterodimeric interaction with Axin(WT). TheAxin(Fu-NT)/Axin(WT) is not conducive to JNK activation, providing a molecular explanation for the dominant negative effect of Axin(Fu-NT) on JNK activation by wild-type Axin. Importantly, Axin(Fu-NT) exhibits no difference in the inhibition of Wnt signaling compared with Axin(WT) as determined by reporter gene assays, interaction with key Wnt regulators, and expression of Wnt marker genes in zebrafish embryos, suggesting that altered JNK signaling contributes, at least in part, to the developmental defects seen in Axin(Fu) mice

Cloning and characterization of G protein beta 1 subunit in shrimp Litopenaeus vannamei

根据G蛋白Gβ1亚基的保守序列设计简并引物,通过简并PCR和RACE技术,克隆到凡纳滨对虾(Litopenaeusvannamei)Gβ1基因的全长cDNA序列。利用Blast、DNAstar和Genedoc软件分析,发现该Gβ1编码的蛋白序列与其他物种已知的Gβ1序列具有相当高的保守性,将它命名为pvGβ1。免疫共沉淀分析发现pvGβ1能在体外适当条件下与对虾Gαs或Gαq相互结合。Westernblot-ting分析发现,pvGβ1在对虾身体各部位都有广泛分布,尤其在脑神经、眼和眼柄有大量表达。说明了Gβ1在对虾的神经系统和光信号传导等生命过程中的重要性。 【英文摘要】 The β/γ-subunits of the heterotrimeric GTP-binding proteins are important regulators of G-protein alpha subunits as well as a series of signal transduction receptors and effectors. In this study, a novel G protein β_1 subunit was isolated from shrimp Litopenaeus vannamei, and was termed pvGβ_1. Sequence analysis showed that the pvGβ_1 protein contained all the well-conserved domains and motifs that were critical sites for interaction with receptors and other binding effectors. Co-immunoprecipitation assay d...国家863计划项目(2002AA629060)资