Shelton Martin N, Huang Ming Bo, Ali Syed, Johnson Kateena, Roth William, Powell Michael, Bond Vincent
Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine.
J Vis Exp. 2013 Jun 30(76):50362. doi: 10.3791/50362.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
从全长蛋白质中发现的基序衍生而来的特定短肽，在我们的研究中是HIV-1 Nef，不仅保留了它们的生物学功能，还能竞争性抑制全长蛋白质的功能。一组20个Nef扫描肽，每个长度为20个氨基酸，且与其相邻肽段重叠10个氨基酸，用于鉴定Nef中负责诱导细胞凋亡的基序。含有这些凋亡基序的肽诱导细胞凋亡的水平与全长Nef蛋白相当。另一种肽，源自Nef的分泌修饰区域（SMR），保留了与外泌体（exNef）中参与Nef分泌的细胞蛋白相互作用的能力。在免疫共沉淀实验中，这种SMRwt肽被用作“诱饵”蛋白，以分离与Nef的SMR基序特异性结合的细胞蛋白。利用蛋白质转染和抗体抑制来物理破坏Nef与分离出的SMR结合蛋白之一mortalin之间的相互作用，并通过基于荧光的exNef分泌测定来测量其效果。SMRwt肽能够在与SMR基序结合的细胞蛋白方面胜过全长Nef，使其成为exNef分泌的首个抑制剂。因此，通过采用本文所述的技术，即利用从全长蛋白质中发现的基序衍生而来的特定短肽的独特性质，人们可以加速蛋白质中功能基序的鉴定以及基于肽的致病功能抑制剂的开发。
