title: Peptide-based identification of functional motifs and their binding partners.
abstract: 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.
authors.affiliation: Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine.
authors.forename: Martin N
authors.initials: MN
authors.lastname: Shelton
authors.forename: Ming Bo
authors.initials: MB
authors.lastname: Huang
authors.forename: Syed
authors.initials: S
authors.lastname: Ali
authors.forename: Kateena
authors.initials: K
authors.lastname: Johnson
authors.forename: William
authors.initials: W
authors.lastname: Roth
authors.forename: Michael
authors.initials: M
authors.lastname: Powell
authors.forename: Vincent
authors.initials: V
authors.lastname: Bond
mesh_terms: D020816:Amino Acid Motifs; D055785:Gene Knockdown Techniques; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; C080352:mortalin; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1