BIO

A driven pre-medical student with a focus on medicine, and 2 years experience in research. Community-geared thinker and team-player. Class of 2025 Presidential Scholar and member of the Phi Eta Sigma National Honor Society.

In the Nick of Time: Understanding the Eviction of an Assembly Chaperone Hsm3 from 26S Proteasomes

Abstract

The proteasome is a complex cellular machine that is responsible for destroying unneeded or unwanted proteins. The proteasome consists of three subcomplexes: the lid, base, and core particle. Each subcomplex can assemble on its own before combining to form mature proteasomes. Under normal conditions, the assembly of the base is controlled by four chaperone proteins. However, how the chaperones are evicted prior to assembly of mature proteasomes is not well understood. We established a transition metal Förster resonance energy transfer (TM-FRET) assay to monitor the eviction of one of these chaperone proteins, Hsm3 from its binding partner in the base, Rpt1. We first produced a single-cysteine version of Hsm3 for labeling at a defined site with the small molecule fluorophore TAMRA. We next engineered two histidines into an α-helix of the base subunit Rpt1 near the Hsm3 binding site to create a Ni2+ chelating site. We performed growth assays on these mutants to confirm that they fully complemented deletion strains, indicating the mutations were innocuous. We will use this TM-FRET system to understand how Hsm3 is evicted during proteasome biogenesis. Proteasome dysfunction has been implicated in several diseases, and is a validated drug target in treating multiple myeloma. Understanding proteasome assembly, and specifically chaperone eviction, can be exploited in developing novel drugs.

Keywords: Medicine, Biochemistry, Microbiology