MalinaLab-Axanton, Tiefenaustr.110, CH-3004 Bern, Switzerland.
J Biomed Sci. 2011 Jan 19;18(1):7. doi: 10.1186/1423-0127-18-7.
The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences.
Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment.
The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.
细胞内的生理过程受可逆的静电蛋白-蛋白相互作用调节。细胞凋亡就是这样一个受调节的过程，它对组织内稳态和发育至关重要，并导致细胞的完全解体。病理性细胞凋亡是一种类似于细胞凋亡的过程，与衰老和感染有关。本研究表明，病理性细胞凋亡是信号蛋白之间的共价相互作用引起的过程，该病理网络的一个特征是钙调蛋白与调节序列的共价结合。
能够共价结合到赖氨酸、组氨酸、精氨酸或谷氨酸的氨基的小分子修饰蛋白质的调节序列。本研究分析了钙调蛋白与 Bax 的 BH3 序列以及原代视网膜上皮细胞培养物和 SV40 转化的鼠上皮成纤维细胞系（野生型[WT]、Bid 敲除[Bid-/-]和 Bax-/-/Bak-/-双敲除[DKO]）中钙调蛋白与豆蔻酰化丙氨酸丰富 C 激酶底物的结合序列。观察到细胞死亡与钙调蛋白的共价结合平行发生，与蛋白质的调节序列结合。黄尿酸已知可激活原代细胞培养物中的 caspase-3，结果表明这种激活也发生在 WT 和 Bid-/-细胞中，但在 DKO 细胞中没有发生。然而，DKO 细胞并未免受死亡的影响，但细胞死亡率很高，导致细胞脱离。
结果表明，小分子修饰蛋白质调节序列中的碱性氨基酸，导致修饰序列（例如钙调蛋白与钙调蛋白结合位点）之间的共价相互作用。这些聚合物（聚集体）的形成导致不受调节的、因此是病理性的蛋白质网络。结果表明小分子参与疾病发展的一种机制。在敲除细胞中，观察到蛋白质之间的不正确相互作用，而没有小分子对蛋白质的修饰，这表明转基因系统中蛋白质网络异常。不可逆的蛋白质-蛋白质相互作用导致蛋白质聚集和细胞退化，这在所有与衰老相关的疾病中都观察到。
