Abstract 1619 The Inhibition of Beta-2-microbglobulin via Novel Bait Peptides

The major histocompatibility complex class 1 (MHC1) is comprised of two proteins, an α chain, and beta-2-microglobulin (β2m). This complex resides on the surface of cells and is important for cellular recognition. When cells undergo apoptosis, the MHC1 is removed from the lipid membrane and expelled into the blood stream. The MHC1 will then enter the kidneys, which allows for the α chain and β2m to be separated. In healthy individuals, the β2m is broken down by the kidney and is excreted out of the body. In patients with renal failure, they require hemodialysis, which leads to a buildup of β2m within the blood stream. This is because hemodialysis machines cannot remove β2m. When β2m builds up within the blood stream, this leads to β2m aggregating with itself, which leads to a condition known as β2m amyloidosis. β2m amyloidosis is responsible for many symptoms such as rheumatoid arthritis, joint destruction, and gastrointestinal defects. By inhibiting β2m aggregation, it is thought that patients undergoing long-term hemodialysis will no longer be affected by β2m amyloidosis, alleviating many symptoms of long-term hemodialysis. To inhibit this aggregation, we have used the MHC1 complex to design multiple bait peptides, which are small proteins that may interfere with β2m aggregation, that are shown to interact with residues of β2m. Specifically, bait peptides were designed around residues Trp60 and His31-Pro32 of β2m, which are known to be important in aggregation. By designing bait peptides that have interactions with these regions, we expect to slow down, or completely inhibit β2m aggregation. With this inhibition, we anticipate that these bait peptides may be used to design treatments in alleviating these destructive side effects of long term hemodialysis. The β2m that will be used in the aggregation assays was expressed and purified over the summer of 2023. Bait peptides were designed using PyMOL and ordered from Genescript. Bait peptides will be resuspended to 1 mg/mL and stored at -20°C once they have arrived. Thioflavin T (ThT) seeded aggregation assays will be run with a fluorescent plate reader, which will quantitate β2m aggregation every 5 minutes, for 10 hours. Using a 94 well plate, different bait peptides at the same concentration will be run in triplicate to quickly assess which ones are best at inhibiting aggregation. From this preliminary data, we well then optimize conditions to determine the maximal effects on aggregation. Alternately, if a single peptide only shows a modest effect on aggregation the combining different peptides to determine if there are additive effect will be explored. Aggregation assays of only β2m will be run as control as well, allowing us to determine how these bait peptides are impacting β2m aggregation. A negative control of ThT in buffer will be run as well as well to make sure nothing in our methods is leading to unexpected aggregation. In the first round of reactions, seeded reactions where used to induce aggregation of β2m. This is when pre-aggregated β2m is added into the well to induce aggregation immediately. However, there was no significant change in the wells with bait peptides compared to the wells without them. This could be due to the interactions between the β2m seeds and the β2m in solution being stronger than β2m to the bait, resulting in normal aggregation. To circumvent this, we have investigated using un-seeded reactions to see if our bait peptides can inhibit the aggregation of β2m. Tri-Beta.