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Immunology and Infection

Peptide-based Identification of Functional Motifs and their Binding Partners

Published: June 30th, 2013

DOI:

10.3791/50362

1Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine, 2Institute for Systems Biology, 3Advanced Medical & Dental Institute, Universiti Sains Malaysia

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef’s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

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.

With the advent of anti-retroviral therapy, the AIDS epidemic in the western world has been slowed, but not curtailed, and the spread of HIV continues to be a major health burden throughout the world. With the exception of the marginally effective RV144 Thai trial, HIV vaccines have so far shown failure to protect from infection. Thus, research into additional, potential therapeutic targets is still warranted.

Along with CD4 T-cell depletion, persistent generalized immune activation is a hallmark of HIV infection. This Chronic Immune Activation (CIA) leads to increases in cell turnover, activated and differentiated lymphocytic subpopulation....

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I. Use of Short Peptides in Biological Analysis

I.1. Mapping Biologically Functional Motifs Using Peptides

  1. Treating cells with Nef scanning peptides
    1. Procure a set of peptides scanning the region of interest. For our experiments, 20mer peptides, with 10 amino acid overlap, spanning the entire HIV-1 Nef protein (205 aa), were obtained from the AIDS Reagent Program. The peptides are assayed individually as follows:
    2. Add 10 ng/ml of peptide to the cell culture medium.......

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Mapping Biologically Functional Motifs using Peptides. Two regions were identified on Nef proteins that induce apoptosis. Peptide-driven apoptosis was observed (Figure 2) beginning with peptide N50 (aa30-50), peaking at N60 (aa40-60) and N70 (aa50-70), and diminishing to background levels at peptide N100 (aa80-100). The major Motif 1 (M1) peak, centering on aa50-60, induces >80% of the apoptotic levels of the full-length Nef protein. A second, smaller apoptotic peak was observed spann.......

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Understanding the mechanisms underlying Nef's ability to manipulate the exosomal trafficking pathway will be useful in engineering novel inhibitors of exNef secretion. Inhibition of exNef secretion should diminish the CD4 T-cell depletion and CIA that drive HIV/AIDS pathogenesis. Towards this goal, we developed a number of novel reagents and methodologies that allowed us to analyze the genetics of exNef secretion, and begin to determine the cellular proteins involved. This approach also led to the development of the firs.......

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This work was supported by NIH/NIGMS/MBRS (Grant 58268), NIH/NCRR/RCMI (Grant G12-RR03034), Georgia Research Alliance funding grant GRA.VAC08.W, NIH/NIAID/NRSA grant F31AI091484, Emory CFAR grant P30 A1050409. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR18386 from NIH/NCRR. The Jurkat cells, the set of 20 HIV-1 Nef peptides, as well as the rabbit anti-HIV-1 Nef antiserum were obtained from the NIH AIDS Research & Reference Reagent Program, (Rockville, MD).

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
20mer peptide set with 10 amino acid overlap NIH AIDS Research and Reference Reagent Program 4641
TUNEL Assay Roche 11 684 809 910
Chariot Protein Delivery Reagent Active Motif 30100
Tecan GENEios fluorimeter (Tecan Group, Switzerland)
96-well black microtiter plate Corning 3792
anti-FLAG M2 Affinity Gel Sigma A2220
Dynabeads Protein G magnetic beads Invitrogen 100.03D
MagnaSphere Technology Magnetic Separation Stand (two position) Promega Corp., Madison, WI Z5332
C-18 ZipTip Millipore ZTC18S096 C18 Resin (0.6 μl or 0.2 μl bed volumes). Oligonucleotides or small (<50 kDa) proteins/ peptides in aqueous solution
MALDI TOF/TOF Bruker Daltonics ultraflex III TOF/TOF

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