Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis

• Published in: Cell 2018-03, Vol.173 (1), p.260-274.e25
• Main Authors: Savitski, Mikhail M., Zinn, Nico, Faelth-Savitski, Maria, Poeckel, Daniel, Gade, Stephan, Becher, Isabelle, Muelbaier, Marcel, Wagner, Anne J., Strohmer, Katrin, Werner, Thilo, Melchert, Stephanie, Petretich, Massimo, Rutkowska, Anna, Vappiani, Johanna, Franken, Holger, Steidel, Michael, Sweetman, Gavain M., Gilan, Omer, Lam, Enid Y.N., Dawson, Mark A., Prinjha, Rab K., Grandi, Paola, Bergamini, Giovanna, Bantscheff, Marcus
• Format: Article
• Language: English
• Subjects: Azepines - chemistry; Azepines - metabolism; Azepines - pharmacology; Cell Line; Chromatography, High Pressure Liquid; Cluster Analysis; degradation; Estradiol - pharmacology; estrogen receptor; estrogen receptors; homeostasis; HSP90; HSP90 Heat-Shock Proteins - metabolism; Humans; Isotope Labeling; JQ1; Jurkat Cells; mass spectrometry; MCF-7 Cells; mechanism of action; Neoplasm Proteins - metabolism; PROTAC; protein degradation; protein turnover; Proteins - antagonists & inhibitors; Proteins - metabolism; proteolysis; Proteolysis - drug effects; proteome; Proteome - analysis; Proteomics - methods; proteostasis; Receptors, Estrogen - metabolism; Tandem Mass Spectrometry; therapeutics; thermal stability; Triazoles - chemistry; Triazoles - metabolism; Triazoles - pharmacology
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2018.02.030

Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed “multiplexed proteome dynamics profiling” (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems. [Display omitted] •Multiplexed proteome dynamics profiling, mPDP, measures changes in proteostasis•JQ1-PROTAC degrades a key mRNA export factor and blocks protein synthesis•Raloxifene induces TMEM97 degradation dysregulating cholesterol homeostasis•Characterization of proteins dependent on HSP90 constitutively or during synthesis Tracking both protein synthesis and degradation across thousands of proteins yields insights into functional regulation by protein degradation.