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This video presents a set of four experiments used for identifying functional HIV one NPH protein motifs and for developing peptide based inhibitors. Specific short peptides derived from motifs found in full length proteins, both retain their biological function and competitively inhibit the function of the full length protein to identify apoptotic motifs. In HIV one NEF jerk cat cells are exposed to NEF scanning peptides and a tunnel apoptotic assay is performed to identify peptides that induce apoptosis.

A second set of peptides containing variations on a NEF secretion motif. The secretion modification region or SMR are transfected into cells along with NPH GFP loss of function due to competitive inhibition indicated by decreased secretion of nph. GFP is assessed by fluorometry to identify cellular factors that interact with the secretion modification region or SMR of nef.

Coun precipitations are performed using SMR wild type NPH as bait. Finally, to test whether these interactions affect secretion, antibodies against identified binding partners are transfected into cells. To assess whether they antagonize NF motif secretion, the results obtained identify two functional apoptotic motifs in NEF and an inhibitor peptide that antagonizes a functional NEF secretion motif as well as its cellular binding partners required for secretion.

The overall goal of this set of four experiments is to accelerate the identification of functional motifs in target proteins and then use that data to develop peptide base inhibitors of these functional motifs. This set of protocols has been useful for our group in helping us to answer key questions related to HIV pathogenesis, such as understanding the role of neph driven secretion in pathogenesis and in deciphering the mechanisms underlying neff's ability to manipulate the exo somal trafficking pathway. Today demonstrating the procedures will be Dr.Ming Bo Huang, who is a research instructor in my laboratory.

Begin this procedure with a set of peptides scanning the region of interest for this experiment. 20 mer peptides with a 10 amino acid overlap spanning the entire HIV one NPH protein were obtained from the AIDS reagent program. To identify NPH apoptotic motifs, perform an apoptosis assay using the NEF scanning peptides individually as follows, add one milliliter of culture medium containing 10 nanograms per milliliter of peptide per 35 millimeter plate of jerk hat, cell cultures, and incubate cultures for 24 hours at 37 degrees Celsius.

Following the incubation, perform a terminal deoxy nucleotide transferase mediated DUTP, biotin nick and labeling or tunnel assay to assess apoptosis. To do this first wash cells with PBS then aspirate the PBS and add 4%Paraform aldehyde in PBS incubate for 30 minutes at room temperature to fix the cells after fixing the cells, wash them with PBS and add Triton X 100, incubate for 10 minutes at room temperature to perme them. After fixing and permeable of cells, add tunnel reagent and incubate the cells for one hour at 37 degrees Celsius following permeable.

Rinse the cells twice with PBS. Then determine the percentage of stained cells by epi fluorescence on a computer controlled microscopy system. After transecting jca cells with NPH, GFP plasmid and different variations of the SMR peptide using chariot protein delivery reagent kit as described in the accompanying document, harvest the medium to determine transfection efficiency by fluorescent microscopy.

Capture fluorescent and brightfield images and determine the percentage of cells. Transfected then to assess inhibition of NPH GFP secretion, transfer 100 microliters of the cell-free conditioned medium to individual wells of a 96 well black microtiter plate. Use a fluorimeter with excitation wavelength 485 nanometers and emission wavelength 515 nanometers to measure the GFP fluorescence in the medium in relative fluorescence units.

After the reading is complete, transfer the data to a pc. Set the level of GFP fluorescence in the control medium at 100%Then compare this to the level of GFP fluorescence in the medium from cells co transfected with various SMR peptides to determine changes in NPH GFP secretion. The co IP for isolating motif binding partners is a difficult step.

Make sure that the beads are properly agitated during incubations and that as much liquid as possible is removed from the beads during each wash Step. Grow jerk hat cells in RPMI 1640 medium containing 10%FBS at 37 degrees Celsius in a T 75 tissue culture flask pellet cells by centrifugation at 1000 times G for 20 minutes at four degrees Celsius. Resuspend the cell pellet in one milliliter of PBS and transfer it to a 1.5 milliliter micro centrifuge tube.

Spin at 1000 times G for one minute. Then resuspend the pellet in one milliliter of Anti-Flag lysis. Buffer by gentle pipetting differentially micro centrifuge.

The suspensions at 2000 times G for five minutes and at 13, 000 times G for 10 minutes. To pellet the cell debris and DNA respectively collect the supernatant hereafter referred to as the lysate. After determining the protein concentration, add one milligram of lysate protein and one microgram of either the SMR wild type or SMR mutant peptide to 20 microliters of Anti-Flag M two affinity gel.

This affinity gel is hereafter referred to as the resin bring the mixture to a final volume of one milliliter in co IP buffer. Also prepare a negative control in which the lysate is combined with resin in the absence of either peptide. Incubate the mixtures at four degrees Celsius overnight with end over end rotation.

The next day pellet the resin by micro centrifugation at 5, 000 to 8, 000 times G for 30 seconds. Following the spin, wait for one to two minutes before handling the samples to allow the resin to settle in the tube. Remove the supernatant with a narrow end pipette tip or a Hamilton syringe taking care not to transfer any resin.

Wash the resin four times by resus, suspending it in 500 microliters of wash buffer, followed by micro centrifugation. To elute the bound cellular proteins. Add 15 micrograms of excess three x flag peptide to the tube in 50 microliters of wash buffer and incubate on a rocker shaker for 30 minutes at room temperature after the incubation centrifuge at 5, 000 to 8, 000 times G for 30 seconds.

After allowing the tubes to settle for two minutes, collect and save the supernatant containing the eluded proteins. After concentrating the EITs by acetone precipitation, boil the samples in laly sample buffer. Then separate the proteins by SDS page on a 40 to 20%triss, HCL criterion precast gel stain the gel with kumasi brilliant blue R two 50.

To assess antibody inhibition cot transfect JCA cells with NGFP and either Antifa tubulin or anti mortality antibody using chariot protein delivery reagent kit as described in the accompanying document, collect the cell-free conditioned medium from the harvested cells. Transfer 100 microliters to individual wells of a 96 well black microtiter plate. Then measure GFP fluorescence in the medium using a fluorimeter with excitation wavelength 485 nanometers and emission wavelength 515 nanometers in relative fluorescent units.

After reading the plate, transfer the data to a PC and set the level of GFP fluorescence in the anti-fat tubulin control at 100%Compare this to the level of GFP fluorescence in the medium from cells cot transfected with anti mortality antibody to determine changes in NEF GFP secretion to map apoptotic motifs of HIV one NPH proteins peptide scanning analysis was performed as described in this video. The Y axis shows percentage of cells that are tunnel labeled and the x axis denotes the treatment condition as shown here, two separate regions of HIV one NEF were identified that induce apoptosis. These are indicated by increased tunnel labeling of cells exposed to neph peptides.

The peaks of apoptosis are denoted by motif one and motif two to assess competitive inhibition of neph secretion by SMR wild type peptides culture medium from jerk CAT T cells cot transected with a NPH GFP clone and various SMR peptides were assay for exo omal NPH secretion as shown by GFP fluorescence in the medium peptides containing one or more wild type NPH SMR sequence motifs inhibited the secretion of exo omal NEF while alanine replacement of any amino acid within this region greatly reduced the peptides effectiveness. This suggests SM R wild type peptide inhibits exo omal nph, an 11 amino acid scrambled version of neff's apoptotic motif. One SM one previously described and obtained from Sigma Genesis was used as a negative control and had no effect on exo omal NEP secretion.

This image of the kumasi stained SDS page gel shows the co immunoprecipitation of four proteins by the SMR wild-type peptide with 60, 65, 75, and 250 kilodalton sizes. The negative control SMR mutant peptide did not capture these proteins, and these proteins did not non-specifically interact with the affinity resin. In the absence of the SMR wild type peptide, these results suggest that the coun precipitated proteins were interacting specifically with the SMR motifs of the SMR wild type peptide.

The plate reader assay shows that cot transfection of the anti mortal antibody with the neph expression plasmid completely abolished exo omal NPH secretion. The unrelated antibody had no effect on exo omal ne secretion. These results suggest that an intact NM talent interaction is required for exo omal neph secretion.

In conclusion, after watching this video, you should have developed a good understanding of how to use the describe techniques to identify functional motifs in target proteins and to use that data to develop peptide base inhibitors of these functional motifs. While attempting the co IP procedure, it will be critical to remember to give careful attention in executing the incubation washes, making sure that the beads are properly agitated during the incubations and that as much liquid as possible is removed from the beads during each wash step. Also during the cell and lysate resin washes always allow the resin to settle following the centrifugation.

Use the narrow end pipette tips and do four washes to adequately reduce the background to acceptable levels.