Developing cross-platform fluorescent reporters for measuring the 26S proteasome and the VCP activities

PROJECT SUMMARY The protein degradation system is closely associated with aging and age-related pathologies. Two master regulators, the 26S proteasome and the VCP/p97 complex, broadly control the ubiquitin-dependent protein breakdown in eukaryotes. Studies of aging and neurodegenerative diseases, such as Alzheimer’s Disease and Related Dementia (AD/ADRDs), frequently involve determining the activities of these regulators, as their activities indicate the progression of aging and neurodegeneration and are important for understanding the underlying molecular mechanisms. Most proteasomal degradation is ubiquitin dependent. This process is tightly regulated by cellular factors and can be affected by disease-associated protein aggregates. Due to the lack of an effective probe for the Ub- dependent 26S proteasome activity in aging and disease-relevant samples, previous studies often employed fluorogenic peptide substrates to measure the Ub-independent peptidase activity of the proteasome. Based on the mechanistic studies by us and others, these peptide substrates are unlikely to detect the actual capacity of the proteasome system in protein degradation and may have contributed to the discrepancies as to the age- dependent changes of proteasome activity in previous studies. The VCP is an Ub-dependent unfoldase that facilitates proteasomal degradation by extracting substrates from membrane and cellular structures and is involved in the pathways, such as ER-associated degradation and ribophagy, which are directly linked to the aging process. The VCP also plays an important role in the clearance of cellular protein aggregates, a hallmark of aging and ADRDs. Despite the broad interest in VCP, it is still challenging to study the role of VCP in aging due to the lack of a specific VCP activity reporter that is applicable in complex biological systems. In this study, we aim to develop a suite of fluorescent reporters to accurately determine the biologically relevant activities of the 26S proteasome and the VCP both in vitro and in vivo. We will validate the performance of these reporters using purified holoenzymes, in tissue/cell extract samples that are commonly involved in the studies of aging and AD/ADRDs and in iPSC-derived neuron cultures. Development of these reporters will significantly benefit the field by enabling applications including: 1). Indicating the progression of aging and age-related diseases; 2). Aiding in understanding the mechanism of the loss of proteostasis during aging and neurodegeneration; 3). Serving as the basis for developing high-throughput screening assays targeting these important enzymes.