Proteasomal Footprinting Reveals the Active Degradome in Peptide Resolution

Introduction:

The mammalian proteasome is estimated to cleave ~70% of all intracellular proteins and is increasingly recognized as a dynamic complex that modulates cellular function in health and disease. However, the regulatory principles targeting specific substrates to proteasomal degradation and their cleavage products are still poorly understood. Current proteomic methods are hindered by the large dynamic range of cellular protein abundance as well as the diversity of modified protein species Moreover, there is no available method for direct capture and analysis of proteasome-cleaved peptides from cells. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows capture, isolation and analysis of proteasome-cleaved peptides[1].

Methods:

To verify whether the isolated peptides were indeed products of proteasomal cleavage, we compared peptides that were identified upon proteasome inhibition to those identified in untreated cells. Likewise, we evaluated whether we could detect the degradation of ZsProSensor, an expected proteasome substrate, using MAPP. Application of MAPP to cancer cell lines as well as primary immune cells reveals dynamic modulation of the cellular degradome in response to various stimuli, such as pro-inflammatory signals. Further, we demonstrate analysis of minute amounts of clinical samples by studying cells from peripheral blood of patients with systemic lupus erythematosus (SLE).

Results:

The peptides identified by MAPP are indeed proteasomally-generated, as they are reduced when the proteasome is inhibited. Furthermore, MAPP retains information on cleavage patterns of proteasomal peptides as the peptides identified had the canonical proteasome cleavage motifs. When examining the rapid changes in the degradome following stimulation with the inflammatory cytokines TNFα and IFNγ we found 8 proteins known to be involved in the cellular response to inflammation are differentially degraded, including HIF1A, ZFAND5 (ZNF216) and CNBP. Finally, we find increased degradation of histones in patient immune cells, which suggests a role for aberrant proteasomal degradation in the pathophysiology of SLE.

Conclusion:

Taken together, MAPP offers a broadly applicable method to facilitate the study of the cellular degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer. The ability to use MAPP in clinical settings with small sample quantities promised to push the forefront of personalized medicine, highlighting new drug targets that are crucial to disease pathogenesis.

[1] Wolf-Levy, H., Javitt, A., Eisenberg-Lerner, A. et al. Revealing the Cellular Degradome by Mass Spectrometry Analysis of Proteasome-Cleaved Peptides. Nature Biotechnology. in Print (2018)