Gaidos Gabriel, Panaitiu Alexandra E, Guo Bingqian, Pellegrini Maria, Mierke Dale F
Chemistry Department, Dartmouth College, Hanover, NH, United States of America.
PLoS One. 2015 Nov 3;10(11):e0141692. doi: 10.1371/journal.pone.0141692. eCollection 2015.
Overexpression of the cellular FLICE-like inhibitory protein (cFLIP) has been reported in a number of tumor types. As an inactive procaspase-8 homologue, cFLIP is recruited to the intracellular assembly known as the Death Inducing Signaling Complex (DISC) where it inhibits apoptosis, leading to cancer cell proliferation. Here we characterize the molecular details of the interaction between cFLIPL and calmodulin, a ubiquitous calcium sensing protein. By expressing the individual domains of cFLIPL, we demonstrate that the interaction with calmodulin is mediated by the N-terminal death effector domain (DED1) of cFLIPL. Additionally, we mapped the interaction to a specific region of the C-terminus of DED1, referred to as DED1 R4. By designing DED1/DED2 chimeric constructs in which the homologous R4 regions of the two domains were swapped, calmodulin binding properties were transferred to DED2 and removed from DED1. Furthermore, we show that the isolated DED1 R4 peptide binds to calmodulin and solve the structure of the peptide-protein complex using NMR and computational refinement. Finally, we demonstrate an interaction between cFLIPL and calmodulin in cancer cell lysates. In summary, our data implicate calmodulin as a potential player in DISC-mediated apoptosis and provide evidence for a specific interaction with the DED1 of cFLIPL.
据报道，细胞FLICE样抑制蛋白（cFLIP）在多种肿瘤类型中存在过表达。作为一种无活性的前半胱天冬酶-8同源物，cFLIP被招募到称为死亡诱导信号复合物（DISC）的细胞内组装体中，在那里它抑制细胞凋亡，导致癌细胞增殖。在这里，我们描述了cFLIPL与钙调蛋白（一种普遍存在的钙传感蛋白）之间相互作用的分子细节。通过表达cFLIPL的各个结构域，我们证明与钙调蛋白的相互作用是由cFLIPL的N端死亡效应结构域（DED1）介导的。此外，我们将这种相互作用定位到DED1 C端的一个特定区域，称为DED1 R4。通过设计DED1/DED2嵌合构建体，其中两个结构域的同源R4区域被交换，钙调蛋白结合特性被转移到DED2并从DED1中去除。此外，我们表明分离的DED1 R4肽与钙调蛋白结合，并使用核磁共振和计算优化解析了肽-蛋白复合物的结构。最后，我们证明了cFLIPL与癌细胞裂解物中的钙调蛋白之间存在相互作用。总之，我们的数据表明钙调蛋白是DISC介导的细胞凋亡中的一个潜在参与者，并为其与cFLIPL的DED1的特异性相互作用提供了证据。
