Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A.
Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A.
复制标题
DOI：
10.3390/ijms21175995
复制
发表时间：
2020-08-20
影响因子：
5.6
通讯作者：
Nikolaidis N
中科院分区：
文献类型：
--
作者：
Smulders L;Daniels AJ;Plescia CB;Berger D;Stahelin RV;Nikolaidis N
HspA1a是一种分子伴侣，可转位到应激和癌细胞的质膜(PM)。这种易位导致HspA1A的细胞表面呈现，从而使肿瘤对辐射不敏感。为了特异性地抑制脂质驱动的HspA1a的PM易位并设计新的治疗方法，必须对未知的HspA1a的脂结合区域进行表征，并确定伴侣与脂结合功能之间的关系。为了阐明这种关系，我们测定了磷脂酰丝氨酸(PS)结合对HspA1A二级结构和伴侣功能的影响。圆二色谱表明，与PS结合后，HspA1a的二级结构发生了极小的变化。无机磷释放的测定表明PS结合对HspA1A的ATPase活性没有影响。相比之下，PS结合显示HspA1A的复性活性略有增加，但持续增加。此外，使用HspA1A的赖氨酸-71-丙氨酸突变(K71A；零ATPase突变体)，我们表明尽管K71A以类似于野生型(WT)的亲和力与PS结合，但突变的蛋白质与脂质结合的速度是WT HspA1A的3倍，解离速度是WT HspA1A的300倍。这些观察表明了一个两步结合模型，包括HspA1A与脂类的初始相互作用，然后是HspA1A-脂类复合体的构象变化，这加速了结合反应。总之，这些发现有力地支持了这样的观点，即HspA1a的伴侣和脂质结合活性是依赖的，但调节这些功能的区域并不重叠，并为未来的干预措施提供了基础，以抑制HspA1a的PM在肿瘤细胞中的移位，使肿瘤细胞对放射治疗敏感。
HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.
登录
查看更多内容
影响因子：
4.6
作者：
Hess, Kyle;Oliverio, Ryan;Nikolaidis, Nikolas
通讯作者：
Nikolaidis, Nikolas
影响因子：
4.2
作者：
Chen, S;Bawa, D;Brown, IR
通讯作者：
Brown, IR
影响因子：
14.8
作者：
Greenfield, Norma J.
通讯作者：
Greenfield, Norma J.
影响因子：
3.4
作者：
Mahalka, Ajay K.;Kirkegaard, Thomas;Kinnunen, Paavo K. J.
通讯作者：
Kinnunen, Paavo K. J.
影响因子：
4.6
作者：
Maurya, Svetlana Rajkumar;Chaturvedi, Deepti;Mahalakshmi, Radhakrishnan
通讯作者：
Mahalakshmi, Radhakrishnan