Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Abstract

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein-protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein-protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein-protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein-protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me 3 )-dependent protein-protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein-protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone ''mark'' appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation ''mark''. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me 3 ). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein-protein interactions mediated by PTMs.

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

“…Further work is needed to determine the full extent to which SAC-MS can illuminate such weak, transient or unstable interactions. For example, it will be interesting to use SAC-MS to investigate enzyme-substrate interactions, a class that is particularly challenging to study by regular affinity capture (90,95,96).…”

Analytical strategies used to identify the readers of histone modifications: A review

“…This approach also confirms survivin is a real H3T3p binder and suggests that MCAK and KIF2A likely interact with H3T3p too [56]. Recently Li group used diazirine as a new photo-reactive group for the identification of HPTMs.…”

Chemical proteomics approaches to examine novel histone posttranslational modifications

“…Furthermore, the application of CLASPI has also enabled the study on the interplay between PTMs at proximal sites of histones. For example, the antagonizing effect of H3K4me3 on survivin binding to H3T3ph was clearly showed by using CLASPI [52]. These studies therefore indicate that CLASPI can be generally applied to identify histone PTM readers and to examine single or multi-PTM-dependent protein-protein interactions.…”

“…In addition, this photo-cross-linking-based approach can also be applied to find readers of other histone methylation marks. For example, it has been demonstrated that human heterochromatin protein-1 (HP1), a known H3K9me3 reader, can be selectively captured by an H3K9me3-based photoaffinity probe (probe 2, Figure 2b) [52]. The same probe was also used for specific labeling of Swi6, the fission yeast homolog of human HP1, in lysates of fission yeast, indicating that this chemical approach can also be used to analyze methylation-dependent protein-protein interactions in a genetically tractable model organism.…”

“…In addition to lysine methylation marks, CLASPI has also been employed to identify readers of phosphorylation on serine (Sph) and threonine (Tph), which are considered as more dynamic and chemically labile PTMs [52]. In the profiling of proteins that recognize H3T3ph, a mitotic histone mark appearing exclusively during cell division, the use of CLASPI resulted in the identification of survivin, the only known H3T3 ph reader, as well as other potential readers such as MCAK and KIF2A.…”