Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and t...Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively. Results demonstrated that mutant a-synuclein resulted in cell death via autophagy and involved a-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant a-synuclein （A30P） also mediated toxicity of 1-methyl-4-phenylpyridinium ion. Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted α-synuclein aggregation and cell death. To further determine the role of autophagy due to mutant α-synuclein, the present study measured expression of microtubule-associated protein light chain 3. Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3 while rapamycin promoted its expression. These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.展开更多
Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun1...Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 （FUNDC1） was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with micro-tubule-associated protein light chain 3 beta （LC3B） through its LC3 interaction region （LIR）. Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDCI LIR peptide phosphorylated at Ser17 （pS17）, demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 （PY18） and Ser13 （PS13） in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry （ITC） assays. Therefore, our structural and biochemical results reveal a working model for thespecific recognition of FUNDCI by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.展开更多
目的探讨β抑制蛋白2（β—arrestin2）及微管相关蛋白轻链3（microtubule—associated protein light chain 3，LC3）在急性肾脏缺血再灌注损伤中的表达及与肾脏损害程度的相关性。方法选用生后3～4周的雄性SD大鼠，随机分为正常组、...目的探讨β抑制蛋白2（β—arrestin2）及微管相关蛋白轻链3（microtubule—associated protein light chain 3，LC3）在急性肾脏缺血再灌注损伤中的表达及与肾脏损害程度的相关性。方法选用生后3～4周的雄性SD大鼠，随机分为正常组、假手术组、急性缺血再灌注损伤组。通过右侧。肾脏切除，无创动脉夹夹闭左侧肾动脉45min之后松开动脉夹，恢复肾脏血流，建立肾脏急性缺血再灌注损伤模型。并在恢复肾脏血流后12、24、36、48、72、96h取肾脏及血液样本。采用免疫组织化学方法及Western blot方法检测各组肾组织中β—arrestin2及LC3蛋白的表达水平，检测各组的肾功能，并对各组肾脏病理学进行评分。结果与正常组及假手术组相比，缺血再灌注损伤组血肌酐及肾脏病理学评分均有显著升高，其中肾脏损伤程度以缺血再灌注损伤后24h最为明显；β—arrestin2及LC3蛋白在正常组及假手术组。肾脏中的表达较少，在缺血再灌注损伤后的肾脏中表达显著升高，其中以缺血再灌注损伤后12h时表达上调最为显著；β—arrestin2及LC3的表达改变先于肾脏病理改变，并且与肾脏损害程度呈正相关（r=0．821，P〈0．05；r=0．913，P〈0．05）。结论在肾脏急性缺血再灌注损伤时，β—arrestin2可能作为一个上游调控蛋白，通过对自噬的调节参与急性。肾损伤的病理过程。展开更多
基金the National Natural Science Foundation of China,No. 30970869a grant from Board of Health of Shanghai,China,No. 2008086+1 种基金Youth Key Project in College of Medicine of Fudan University,No. 09-L37a grant from the Project of Shanghai Key Laboratory of Diabetes Mellitus,No. 08DZ2230200
文摘Several studies have demonstrated that overexpression of mutant a-synuclein in PC12 cells is related to occurrence of autophagy. The present study established mutant α-synuclein （A30P） -transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively. Results demonstrated that mutant a-synuclein resulted in cell death via autophagy and involved a-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant a-synuclein （A30P） also mediated toxicity of 1-methyl-4-phenylpyridinium ion. Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted α-synuclein aggregation and cell death. To further determine the role of autophagy due to mutant α-synuclein, the present study measured expression of microtubule-associated protein light chain 3. Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3 while rapamycin promoted its expression. These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.
基金This work was supported by National Natural Science Founda- tion （Grant No. 31400629） the Strategic Priority Research Program of the Chinese Academy of Science （No. XDB08010101）+1 种基金 Ministry Of Science And Technology of China （No. 2016YFA0500700） China Postdoctoral Science Foundation （No. 2015M582009 and 2016T90579） and National Natural Science Foundation （Grant No. 31330018）.
文摘Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 （FUNDC1） was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with micro-tubule-associated protein light chain 3 beta （LC3B） through its LC3 interaction region （LIR）. Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDCI LIR peptide phosphorylated at Ser17 （pS17）, demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 （PY18） and Ser13 （PS13） in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry （ITC） assays. Therefore, our structural and biochemical results reveal a working model for thespecific recognition of FUNDCI by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.
