AMPylation Signaling Participates in Diverse Processes with Pleiotropic Actions

Truttmann MC et al.[1] recently reported that AMPylation of heat shock protein 70 (HSP70) family of chaperones participates in altering the aggregation properties and maintaining protein homeostasis (proteostasis), thereby playing a vital role in the development of neurodegenerative diseases (NDs). NDs are commonly manifested by protein aggregates, which exert harmful effects on proteostasis. Interestingly, it has been observed that AMPylation of heat shock proteins (HSPs) can maintain proteostasis by inhibiting the formation of protein aggregates. As previous studies only indicate that HSPs could regulate proteostasis, such a novel discovery further demonstrates the involvement of HSP70 AMPylation in the regulation of protein aggregation and the maintenance of proteostasis. Therefore, AMPylation can be considered to possess a therapeutic potential to target certain physiological processes related to proteostasis, such as age-related diseases.

鼠伤寒沙门菌修饰酶YdiU的表达、纯化及晶体培养

目的采用大肠埃希菌原核表达体系表达并纯化YdiU蛋白,筛选蛋白质晶体,为其结构和酶活测定等研究奠定基础。方法以生物信息学预测为基础,使用PCR方法从沙门菌基因组中调取ydiU基因,构建ydiU-pGl01重组质粒,转化入大肠埃希菌BL21(DE3)中,诱导蛋白表达。并使用镍离子亲和层析柱和阴离子交换柱对YdiU蛋白进行纯化。纯化后YdiU蛋白利用16种晶体筛选试剂盒在悬滴条件下进行晶体培养。结果成功构建重组表达质粒ydiU-pGl01。IPTG诱导后,YdiU蛋白在大肠埃希菌BL21(DE3)菌株中大量表达,并多以可溶形式存在。经镍离子亲和层析和阴离子交换法纯化后得到纯度较高的蛋白溶液,浓度为3.6 mg/ml,相对分子质量约51×10^3。纯化的YdiU蛋白在0.5 mol/L Potassium thiocyanate条件下长出蛋白晶体。结论采用大肠埃希菌表达系统可表达可溶性YdiU蛋白,YdiU蛋白在阴离子交换柱上表现为峰型对称的单峰,纯度较高,可用于蛋白质晶体的筛选,为进一步解析YdiU的结构和功能研究奠定了基础。