Thimet oligopeptidases (TOPs) are metallopeptidases that are widely distributed in the Animal and Plant kingdoms that are involved in maintaining cellular proteostasis. Although the two major TOPs members in plants have high sequence homology, TOP1 is located within chloroplasts and mitochondria and TOP2 is found within the cytosol. TOPs have been shown in <i>Arabidopsis thaliana</i> to be critical constituents in plant responses to pathogens, but a deeper description of TOPs and the accompanying peptide substrates is required to characterize their contributions biochemical signaling during plant stress events. A multitude of potential TOPs substrates have been identified from a variety of different processes in the cell such as photosynthesis, protein folding, and glycolysis. Variety in TOPs substrates indicates functionality beyond plant defense which can be used to study diverse signaling mechanisms within plant cells. Potential TOPs substrates are identified using peptidomic methods via liquid chromatography-tandem mass spectrometry (LC-MS/MS), synthesized using Fmoc-based semi-automated solid-phase peptide synthesis (SPPS), and purified using preparative high-performance liquid chromatography (HPLC). These peptide substrates are then tested for TOPs activity through <i>in vitro</i> proteolytic digestion and LC-MS/MS analysis. By characterizing the selectivity of TOPs proteolytic activity and their roles in plant defense and signaling, a clearer understanding of plant responses to biotic and abiotic stresses can be established.

Synthesis of thimet oligopeptidase (TOP) substrates for analysis of plant defense mechanisms

ACS Spring 2022

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