Biologically distinct conformations of Bcl-x can be resolved using 2D isoelectric focusing.
Biologically distinct conformations of Bcl-x can be resolved using 2D isoelectric focusing.
复制标题
Bcl-x 的生物学独特构象可以使用 2D 等电聚焦来解析。
DOI：
10.1016/j.molimm.2009.02.031
复制
发表时间：
2009
影响因子：
3.6
通讯作者：
Huber,BrigitteT
中科院分区：
文献类型：
--
作者：
Rockwell,KarenR;Huber,BrigitteT
Bcl-x是细胞决定生死的一种有效调节因子，它依赖于不同的蛋白质区域，具有多种增强生存的活性。有证据表明，根据局部环境和蛋白质或肽伴侣的结合，Bcl-x可以呈现几种构象，暴露不同的蛋白质区域。然而，构象形式的生物学发生一直很难研究，因为结构测定技术使用大量的蛋白质，在改变Bcl-x构象的条件下纯化。我们在这里表明，标准的二维等电聚焦技术可以用来区分细胞裂解物中的Bcl-x的构象不同的形式。通过使用不同pH值的洗涤剂和缓冲液来操纵构象等电形式。我们的数据表明，翻译后修饰不需要或与构象变化相关，区分Bcl-x的主要等电形式。我们发现，Bcl-x构象等电形式具有优选的亚细胞定位模式。此外，在IL-3依赖性细胞的细胞因子饥饿期间，构象形式在某些位置受到不同的调节。因此，我们提供的证据表明，2DIEF可以用来查看生物学上不同的构象差异Bcl-x对微量的未纯化的蛋白质从细胞或裂解物。
Bcl-x, a potent regulator of cellular decisions of life and death, has multiple survival-enhancing activities that rely on distinct protein regions. Evidence suggests that depending on the local environment and the binding of protein or peptide partners, Bcl-x can take on several conformations that expose different protein regions. However, biological occurrence of conformational forms has been very difficult to study, because structure determination techniques use large quantities of protein, purified under conditions that change Bcl-x conformation. We show here that standard 2D isoelectric focusing techniques can be used to distinguish conformationally distinct forms of Bcl-x in cell lysates. Conformational isoelectric forms were manipulated through the use of detergents and buffers of differing pH. Our data indicate that post-translational modifications are not needed for or associated with conformational changes, distinguishing the dominant isoelectric forms of Bcl-x. We found that Bcl-x conformational isoelectric forms have preferred subcellular localization patterns. Moreover, conformational forms are differently regulated in certain locations during cytokine starvation of IL-3-dependent cells. Therefore, we provide evidence that 2DIEF can be used to view biologically distinct conformational differences in Bcl-x on minute quantities of unpurified protein from cells or lysates.