Abstract
Post-translational modification (PTM) crosstalk is recognized as a major cell-regulatory mechanism, and studies of several proteins have validated the premise that PTMs work in concert. Previous work by our group investigated the potential PTM crosstalk on proteins in the EGFR-Ras-c-Fos axis by utilizing a comprehensive set of PTM reagents termed Signal-Seeker toolkits. In this study, these tools were used to investigate the potential PTM crosstalk that occurs in acetylated mitochondrial proteins in response to a mitochondrial stress-inducing agent hydrogen peroxide (H2O2). Mitochondrial protein acetylation has been shown to participate in PTM crosstalk as exemplified by the regulation of the pyruvate dehydrogenase complex via kinase, phosphatase, acetyltransferase, and deacetylase activities. Changes in the acetylated state of mitochondrial proteins were investigated, in response to H2O2, using a novel anti acetyl lysine (Ac-K) antibody. Signal-Seeker PTM detection tools were used to validate the acetylation state of ten mitochondrial targets, as well as their endogenous acetylation state in response to H2O2. Importantly, the endogenous acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation state of four target mitochondrial proteins were also investigated with the toolkit. Each of the four proteins had unique PTM profiles, but diverging acetylation and ubiquitin or SUMO 2/3 signals appeared to be a common theme. This proof-of-concept study identifies the Signal-Seeker toolkits as a useful tool to investigate potential PTM crosstalk.
Highlights
Post-translational modifications (PTMs) are dynamic, tightly-regulated alterations to a protein that control the protein’s structure, spatial localization, and interactions; thereby, governing its function [1,2,3]
Shows convincing co-localization of AAC02 and mitochondrial structures, but minimal overlap with vesicles. While this data suggests that AAC02 identifies acetylated mitochondria proteins, it does not determine whether AAC02 can detect dynamic and physiologic changes of acetylated proteins in the mitochondria
For example phosphorylation has been shown to be a precursor to target protein Ub in several proteins [46,47]. Another pattern of PTM crosstalk exists between Ac and Ub where these modifications have been reported to compete to regulate the stability of the target protein [48,49]
Summary
Post-translational modifications (PTMs) are dynamic, tightly-regulated alterations to a protein that control the protein’s structure, spatial localization, and interactions; thereby, governing its function [1,2,3]. Well-studied PTMs include, acetylation (Ac), ubiquitination (Ub), small ubiquitin-like modifier 2/3 (SUMOylation 2/3 (SUMO 2/3)), and tyrosine phosphorylation (pY), but there are many others [4,5,6,7,8]. Unbiased approaches such as mass spectrometry (MS)-proteomics have championed the growth of the PTM field by unveiling the vast abundance of PTM targets [9]. Recent PTM studies of Proteomes 2018, 6, 24; doi:10.3390/proteomes6020024 www.mdpi.com/journal/proteomes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
