摘要
本研究主要从蛋白质结构分析Akt1 SUMO化的位点及位点的突变对其结构与功能的影响。采用多种软件分析Akt1 SUMO化位点和Akt1野生型(Akt1wt)及Akt1K64/276R的理化性质、亲/疏水性及二/三级结构;分析结果显示,Akt1K64/276R较Akt1wt,亲/疏水性未改变,α-螺旋和β-折叠都有少量的不同。三级结构分析显示,与野生型组相比,Akt1K64R氢键增多。以Myc-Akt1wt-pcDNA3.1为模板,采用PCR定点突变技术扩增出Myc-Akt1K64/276R。DNA序列分析结果显示,Myc-Akt1K64/276R基因序列编码赖氨酸(K)的密码子AAG被成功突变为精氨酸(R)密码子AGG。免疫沉淀和免疫印迹结果显示,不共转PIAS3,Akt1也能与SUMO1结合;Myc-Akt1wt和Myc-Akt1K64/276R均可在HEK293细胞中高效表达;转染Myc-Akt1K64/276R组SUMO化水平降低了70%左右(P<0.05)。免疫印迹结果显示,在小鼠海马神经细胞HT22中,Myc-Akt1wt组ERK1/2磷酸化水平及BDNF蛋白水平是突变组的约1.5倍(P<0.05);野生型组p-Elk1是突变体组的2倍(P<0.05),而mTOR、P70S6K、4E-BP1的表达及磷酸化均无显著改变。以上结果表明,Akt1中K64/276的突变对蛋白质结构和表达未见影响,仅引起Akt1 SUMO化降低及下游ERK1/2-Elk1-BDNF信号通路的抑制。
In this study, the SUMO(small ubiquitin-like modifier)-modified sites of Akt1(protein kinase Bα) and the effect of SUMOylation deficiency on the structure and function of Akt1 are analyzed. The online software analyses were used to verify the SUMOylation sites of Akt1, and the physicochemical properties, hydrophilicity/hydrophobicity, and secondary/tertiary structure of the wild-type Akt1(Akt1 wt) and Akt1 mutant(Akt1 K64/276 R). The bioinformatic analysis results revealed that the hydrophilic/hydrophobic properties of Akt1 K64/276 R were not changed compared with Akt1 wt, but the numbers of α-helix and β-sheet were slightly different. The tertiary structure analysis showed that the hydrogen bonds increased in Akt1 K64/276 R compared to Akt1 wt. The Myc-Akt1 K64/276 R-pcDNA3.1 was constructed by PCR site-directed mutagenesis using Myc-Akt1 wt-pcDNA3.1 as templates. DNA sequence analysis showed that the AAG coding lysine(K) in Akt1 was successfully mutated to the arginine(R) codon AGG. IP(immunoprecipitation) and IB(immunoblotting) results showed that Akt1 can also conjugate with SUMO1 without PIAS3(protein inhibitor of activated STAT 3).Myc-Akt1 wt and Myc-Akt1 K64/276 R were both highly expressed in HEK293 cells. Compared with Myc-Akt1 wt, SUMO1 ylation levels were significantly reduced about 70% in Akt1 K64/276 R. In mouse hippocampal neurons HT22 cells, phosphorylation and expression of ERK1/2, Elk1, mTOR, P70 S6 K and 4 E-BP1, and the BDNF protein expression were examined by IB. ERK1/2 phosphorylation and BDNF protein expression in the Myc-Akt1 wt group were about 1.5 times higher than those in the mutant group(P < 0.05). The expression and phosphorylation of mTOR, P70 S6 K and 4 E-BP1 in the wild-type and mutation group displayed no significant changes. In conclusion, the mutation of K64/276 in Akt1 has no effect on protein structure and expression, but only causes the reduction of Akt1 SUMOylation and inhibition of ERK1/2-Elk1-BDNF cascades.
作者
孟利
杜彩萍
MENG Li;DU Cai-Ping(Research Center for Biochemistry and Molecular Biology,Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou Medical University,Xuzhou 221004,Jiangsu,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2022年第12期1661-1670,共10页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.81100852)
江苏省高等学校自然科学研究重大项目(No.20KJA310010)资助。
