Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using Limited Proteolysis

Conformational changes in proteins
can lead to disease. Thus, methods
for identifying conformational changes in proteins can further improve
our understanding and facilitate detection of disease states. Here
we combine limited proteolysis (LiP) with Stable Isotope Labeling
with Amino Acids in Cell Culture (SILAC) to characterize breast cancer-related
conformational changes in proteins on the proteomic scale. Studied
here are the conformational properties of proteins in two cell culture
models of breast cancer, including the MCF-10A and MCF-7 cell lines.
The SILAC-LiP approach described here identified ∼200 proteins
with cell-line-dependent conformational changes, as determined by
their differential susceptibility to proteolytic digestion using the
nonspecific protease, proteinase K. The protease susceptibility profiles
of the proteins in these cell lines were compared to thermodynamic
stability and expression level profiles previously generated for proteins
in these same breast cancer cell lines. The comparisons revealed that
there was little overlap between the proteins with protease susceptibility
changes and the proteins with thermodynamic stability and/or expression
level changes. Thus, the large-scale conformational analysis described
here provides unique insight into the molecular basis of the breast
cancer phenotypes in this study.

蛋白质的构象变化可引发疾病。因此，可识别蛋白质构象变化的方法，能够进一步加深我们对疾病的认知，并助力疾病状态的检测。本研究将有限蛋白水解法（limited proteolysis, LiP）与细胞培养氨基酸稳定同位素标记法（Stable Isotope Labeling with Amino Acids in Cell Culture, SILAC）相结合，在蛋白质组层面上表征与乳腺癌相关的蛋白质构象变化。本研究聚焦两种乳腺癌细胞培养模型中的蛋白质构象特性，所涉及的细胞系包括MCF-10A与MCF-7。本研究采用的SILAC-LiP联用策略，通过非特异性蛋白酶K的蛋白水解消化实验，基于蛋白质消化敏感性的差异，鉴定出约200种存在细胞系依赖性构象变化的蛋白质。研究人员将这些细胞系中蛋白质的蛋白酶敏感性谱图，与此前针对同一批乳腺癌细胞系蛋白质所获得的热力学稳定性及表达水平谱图进行了对比。对比结果显示，存在蛋白酶敏感性变化的蛋白质，与存在热力学稳定性及/或表达水平变化的蛋白质之间重合度极低。因此，本研究中采用的大规模构象分析方法，为解析本次研究中乳腺癌表型的分子机制提供了独特视角。