Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Protein TAILS tell remarkable tales: Positional proteomics reveals diverse N-Terminomes and proteolytic landscapes in disease (#29)

Chris Overall 1
  1. University of British Columbia, Vancouver, BC, Canada

Identification of protein terminal peptides provides key information on protein stability and function. Our degradomics methods enrich and annotate terminomes—Terminal Amine Isotopic Labelling of Substrates (TAILS, Kleifeld etal 2010), Amino Terminal Orientated Mass Spectrometry (ATOMS), Proteome-wide Identification of Protease Cleavage Specificity (PICS, Schilling_&_Overall 2008), LysargiNase, the new protease for proteomics we discovered (Huesgen etal 2015), and our N and C-termini database version 4.0 TopFIND (http://clipserve.clip.ubc.ca/topfind) utilised in our studies reveal widespread truncation and generation of termini in normal and diseased tissues.

Certain N terminal semi-tryptic peptides exhibit beneficial m/z, ionization and fragmentation properties over tryptic peptides, rendering these peptides and proteins identifiable. In the C-HPP, TAILS was used to identify proteoforms and also provide MS evidence for the expression of PE2-4 ‘missing proteins’, in rare tissues and cells.

We discovered active protease domains in bacterial flagella of >200 species with ~1,000 cleavage sites for “flagellinolysin” being identified by PICS. With ~20,000 flagellin copies/~10-μm flagella this assembles the largest proteolytic complex known with potential for numerous roles in saprophytic bacteria and in pathogens.

ATOMS was used to identify function-modifying cleavages by metalloproteinases in moonlighting extracellular tRNA synthetases. When moonlighting outside the cell, “intracellular” tyrosyl tRNA synthetase was proteolytically activated as a proinflammatory mediator signaling through Toll-like receptor-2 (TLR2), resulting in NF-κB activation and TNα/chemokine release from macrophages. In tryptophan tRNA synthetase, cleavage inactivated TLR signalling and proinflammatory functions.

 By TAILS we previously reported that the innate immune cell MMPs orchestrate leukocyte chemotaxis by cleavage of most human chemokines. More recently IFNα and IFNγ, and complement proteins were discovered as new MMP substrates. MMP processing of these bioactive substrates dampens inflammation essential for terminating inflammatory responses and inspiring new potential novel precision therapies to modulate protease enzyme activities in diseases due to protease deregulation.