Revealing functional insights into ER proteostasis through proteomics and interactomics

Abstract

The endoplasmic reticulum (ER), responsible for processing approximately one-third of the human proteome including most secreted and membrane proteins, plays a pivotal role in protein homeostasis (proteostasis). Dysregulation of ER proteostasis has been implicated in a number of disease states. As such, continued efforts are directed at elucidating mechanisms of ER protein quality control which are mediated by transient and dynamic protein-protein interactions with molecular chaperones, co-chaperones, protein folding and trafficking factors that take place in and around the ER. Technological advances in mass spectrometry have played a pivotal role in characterizing and understanding these protein-protein interactions that dictate protein quality control mechanisms. Here, we highlight the recent progress from mass spectrometry-based investigation of ER protein quality control in revealing the topological arrangement of the proteostasis network, stress response mechanisms that adjust the ER proteostasis capacity, and disease specific changes in proteostasis network engagement. We close by providing a brief outlook on underexplored areas of ER proteostasis where mass spectrometry is a tool uniquely primed to further expand our understanding of the regulation and coordination of protein quality control processes in diverse diseases.

Keywords: Affinity purification-mass spectrometry; Endoplasmic reticulum; Mass spectrometry; Protein quality control; Unfolded protein response.

Figure 1.. Secretory proteostasis and protein quality control is governed by the activity of ER protein folding, trafficking, and degradation pathways.

Proteins routed for secretory environments cotranslationally enter the ER and engage with chaperones and other folding factors or enzymes that aid in client protein folding. Upon reaching a properly folded confirmation, client proteins are packaged and targeted towards secretory environments through engagement with vesicle trafficking machinery. Conversely, client protein unable to reach their proper conformation are routed towards degradation pathways such ERAD or autophagy (ER-phagy). Arrows denoted in the figure are meant to exemplify interactions with different protein folding pathways of the ER. Routing is highly client-specific and a given protein client may not engage with all of the proteostasis factors and pathways.

Figure 2.. ER protein quality control capacity in metazoans is monitored by the three branches of the unfolded protein response.

The UPR is regulated by three signaling branches consisting of IRE1, ATF6, and PERK. Yeast lack the ATF6 pathway and only contain the most evolutionarily conserved IRE1 and PERK branches.

Figure 3.. Affinity purification – mass spectrometry and tool compounds to elucidate protein-protein interactions implicated in protein quality control.

A. Schematic detailing a typical AP-MS workflow. B. Proximity labeling MS often uses a biotin ligase-tagged protein of interest to biotinylate interacting proteins. Biotinylated proteins can then be affinity purified and analyzed via LC-MS/MS to identify interacting partners. C. Protein crosslinkers can be used to aid in affinity purification of interacting proteins by covalently linking them and can subsequently be used to identify crosslinked peptides in some cases. D. Stable isotope labeling with amino acids has aided in quantitative mass spectrometry experiments. Blue asterisks indicate atoms where heavy isotopes can be incorporated. E. Isobaric labeling has additionally aided in quantitative mass spectrometry investigation as well as increased throughput through multiplexing. Blue asterisks indicate points where heavy isotopes can be incorporated.