Effect of Protein Denaturation and Enzyme Inhibitors on Proteasomal-Mediated Production of Peptides in Human Embryonic Kidney Cells.

Sayani Dasgupta,Leandro M Castro,Alexandre K Tashima,Emer S Ferro,Michael A Fishman,Lloyd D Fricker

doi:10.3390/biom9060207

Abstract

Peptides produced by the proteasome have been proposed to function as signaling molecules that regulate a number of biological processes. In the current study, we used quantitative peptidomics to test whether conditions that affect protein stability, synthesis, or turnover cause changes in the levels of peptides in Human Embryonic Kidney 293T (HEK293T) cells. Mild heat shock (42 °C for 1 h) or treatment with the deubiquitinase inhibitor b-AP15 led to higher levels of ubiquitinated proteins but did not significantly increase the levels of intracellular peptides. Treatment with cycloheximide, an inhibitor of protein translation, did not substantially alter the levels of intracellular peptides identified herein. Cells treated with a combination of epoxomicin and bortezomib showed large increases in the levels of most peptides, relative to the levels in cells treated with either compound alone. Taken together with previous studies, these results support a mechanism in which the proteasome cleaves proteins into peptides that are readily detected in our assays (i.e., 6–37 amino acids) and then further degrades many of these peptides into smaller fragments.

Highlights

Mass spectrometry-based quantitative peptidomic techniques were initially developed to identify neuropeptides and peptide hormones in a variety of organisms [1,2,3,4]
To investigate whether this pathway is required for the production of intracellular peptides, we treated cells with b-AP15 which inhibits two of the DUBs that are transiently associated with the 19S regulatory particle: ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14) [32,34]
To investigate whether b-AP15 affects the levels of intracellular peptides, Human Embryonic Kidney 293T (HEK293T) cells were treated with either 1 μM b-AP15 or a comparable concentration of dimethyl sulfoxide (DMSO) for 1 h and the relative levels of cellular peptides were measured using a quantitative peptidomics approach

Summary

Introduction

Mass spectrometry-based quantitative peptidomic techniques were initially developed to identify neuropeptides and peptide hormones in a variety of organisms [1,2,3,4]. In addition to detecting a large number of neuropeptides and other peptides derived from secretory pathway proteins, peptidomic studies detected hundreds of peptides derived from cytosolic, nuclear, and mitochondrial proteins [5]. Some of these intracellular peptides may play a role in biological functions by regulating protein–protein interactions, much like synthetic peptides modulate cellular functions by mimicking or blocking protein–protein interactions [5,6,7]. Svb transcriptional repressor into a shorter activator [9] Taken together, these studies suggest that intracellular peptides play important roles in regulating cellular functions

Methods

Results

Conclusion