p.G38R和p.D40G突变通过改变ANXA11蛋白亚细胞定位引发肌萎缩侧索硬化症
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摘要
目的本研究旨在研究Annexin A11蛋白(ANXA11)N端突变p.G38R和p.D40G对该蛋白亚细胞定位的影响,探索其引起肌萎缩侧索硬化症(ALS)的机制。方法构建表达ANXA11野生型蛋白(wt)及p.G38R和p.D40G突变蛋白的真核表达载体,采用激光共聚焦显微镜检测3种蛋白在HEK293细胞中的亚定位。结果 ANXA11wt、p.G38R及p.D40 G蛋白均可在HEK 293细胞中形成囊泡状结构,其中p.G38 R组囊泡面积大于wt组(P=0.002)和p.D40 G组(P=0.006),后两组间无明显差异(P=0.791)。在单个细胞中,ANXA11 wt形成的囊泡数目大于p.G38 R组(P=0.002)和p.D40 G组(P<0.001),而p.G38 R组和p.D40 G组差异不明显(P=0.516)。与ANXA11 wt相比,p.G38 R蛋白(P<0.001)和p.D40 G蛋白(P<0.001)在细胞核内的分布显著减少,后两者间无明显差异(P=0.519)。结论 p.G38 R和p.D40 G突变减少了ANXA11蛋白相关囊泡状结构的数目和ANXA11蛋白在细胞核的分布,这可能是引起ALS发病的机制之一。
Objective To investigate the influence of the N-terminal mutations of Annexin A11( ANXA11) protein,p. G38 R and p.D40 G,on the subcellular localization of this protein and their mechanism of action in inducing amyotrophic lateral sclerosis( ALS).Methods Eukaryotic expression vectors which expressed ANXA11 wild-type protein,ANXA11 p. G38 R protein,or ANXA11 p. D40 G protein were constructed,and a laser scanning confocal microscope was used to investigate the subcellular localization of these three proteins in HEK293 cells. Results ANXA11 wild-type protein,ANXA11 p. G38 R protein,and ANXA11 p. D40 G protein formed a vesicular structure in HEK293 cells,and the p. G38 R group had significantly larger vesicles than the ANXA11 wild-type group( P =0. 002) and the p. D40 G group( P = 0. 006),while there was no significant difference between the latter two groups( P = 0. 791). In a single cell,the ANXA11 wild-type group had significantly more vesicles than the p. G38 R group( P = 0. 002) and the p. D40 G group( P < 0. 001),and there was no significant difference between the p. G38 R group and the p. D40 G group( P = 0. 516). Compared with ANXA11 wild-type protein,p. G38 R protein and p. D40 G protein had significantly reduced distribution in the nucleus( both P <0. 001),and there was no significant difference between p. G38 R protein and p. D40 G protein( P = 0. 519). Conclusions The p.G38 R and p. D40 G mutations reduce the number of vesicles of ANXA11 protein and the distribution of ANXA11 protein in the nucleus,which may be one of the mechanism of ALS.
Objective To investigate the influence of the N-terminal mutations of Annexin A11( ANXA11) protein,p. G38 R and p.D40 G,on the subcellular localization of this protein and their mechanism of action in inducing amyotrophic lateral sclerosis( ALS).Methods Eukaryotic expression vectors which expressed ANXA11 wild-type protein,ANXA11 p. G38 R protein,or ANXA11 p. D40 G protein were constructed,and a laser scanning confocal microscope was used to investigate the subcellular localization of these three proteins in HEK293 cells. Results ANXA11 wild-type protein,ANXA11 p. G38 R protein,and ANXA11 p. D40 G protein formed a vesicular structure in HEK293 cells,and the p. G38 R group had significantly larger vesicles than the ANXA11 wild-type group( P =0. 002) and the p. D40 G group( P = 0. 006),while there was no significant difference between the latter two groups( P = 0. 791). In a single cell,the ANXA11 wild-type group had significantly more vesicles than the p. G38 R group( P = 0. 002) and the p. D40 G group( P < 0. 001),and there was no significant difference between the p. G38 R group and the p. D40 G group( P = 0. 516). Compared with ANXA11 wild-type protein,p. G38 R protein and p. D40 G protein had significantly reduced distribution in the nucleus( both P <0. 001),and there was no significant difference between p. G38 R protein and p. D40 G protein( P = 0. 519). Conclusions The p.G38 R and p. D40 G mutations reduce the number of vesicles of ANXA11 protein and the distribution of ANXA11 protein in the nucleus,which may be one of the mechanism of ALS.
引文
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[2]Chen L,Zhang B,Chen R,et al.Natural history and clinical features of sporadic amyotrophic lateral sclerosis in china[J].J Neurol Neurosurg Psychiatry,2015,869(10):1075-1081.
[3]Zufiria M,Gil-Bea FJ,Fernandez-Torron R,et al.Als:Abucket of genes,environment,metabolism and unknown ingredients[J].Prog Neurobiol,2016,142:104-129.
[4]Nguyen HP,Van Broeckhoven C,van der Zee J.Als genes in the genomic era and their implications for ftd[J].Trends Genet,2018,34(6):404-423.
[5]Smith BN,Topp SD,Fallini C,et al.Mutations in the vesicular trafficking protein annexin all are associated with amyotrophic lateral sclerosis[C].Sci Transl Med,2017,9(388):9157.
[6]Gerke V,Moss SE.Annexins:From structure to function[J].Physiol Rev,2002,82(2):331-371.
[7]Lecona E,Turnay J,Olmo N,et al.Structural and functional characterization of recombinant mouse annexin all:Influence of calcium binding[J].Biochem J,2003,373(Pt 2):437-449.
[8]Wang J,Guo C,Liu S,et al.Annexin all in disease[J].Clin Chim Acta,2014,431:164-168.
[9]Satoh H,Nakano Y,Shibata H,et al.The penta-ef-hand domain of alg-2 interacts with amino-terminal domains of both annexin vii and annexin xi in a ca2+-dependent manner[J].Biochim Biophys Acta,2002,1600(1-2):61-67.
[10]Williams LH,McClive PJ,Van Den Bergen JA,et al.Annexin xi co-localises with calcyclin in proliferating cells of the embryonic mouse testis[J].Develop Dynam,2005,234(2):432-437.
[11]Shibata H,Kanadome T,Sugiura H,et al.A new role for annexin a11 in the early secretory pathway via stabilizing sec31 a protein at the endoplasmic reticulum exit sites(eres)[J].J Biol Chem,2015,290(8):4981-4993.
[12]Lloyd-Lewis B,Krueger CC,Sargeant TJ,et al.Stat3-mediated alterations in lysosomal membrane protein composition[J].J Biol Chem,2018,293(12):4244-4261.
[13]Mizutani A,Watanabe N,Kitao T,et al.The long aminoterminal tail domain of annexin xi is necessary for its nuclear localization[J].Arch Biochem Biophys,1995,318(1):157-165.
[14]Farnaes L,Ditzel HJ.Dissecting the cellular functions of annexin xi using recombinant human annexin xi-specific autoantibodies cloned by phage display[J].J Biol Chem,2003,278(35):33120-33126.
[15]Baltz AG,Munschauer M,Schwanhausser B,et al.The mrna-hound proteome and its global occupancy profile on protein-coding transcripts[J].Mol Cell,2012,46(5):674-690.