TY - JOUR
T1 - A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells
AU - Kleiner, Ralph E.
AU - Hang, Lisa E.
AU - Molloy, Kelly R.
AU - Chait, Brian T.
AU - Kapoor, Tarun M.
N1 - Funding Information:
We are grateful to Peter Schultz for the generous gift of amber suppression plasmid pCMV-AbK. R.E.K. acknowledges support from the Charles H. Revson Foundation through a Revson Senior Fellowship in Biomedical Science. L.E.H. was supported by NRSA Training grant no. CA009673-37 . The authors acknowledge support from the US NIH ( GM98579 to T.M.K., PHS GM103314 and PHS GM109824 to B.T.C.).
Publisher Copyright:
© 2017 Elsevier Ltd
PY - 2018/1/18
Y1 - 2018/1/18
N2 - Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics. Protein-protein interactions mediate essential biological processes, but characterizing these interactions in cells presents a major challenge. Kleiner et al. describe a chemical proteomics approach combining photo-crosslinking and SILAC-based proteomics to characterize context-dependent direct protein-protein interactions in live cells. They apply their approach to profile interactions involving core histones H3 and H4.
AB - Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics. Protein-protein interactions mediate essential biological processes, but characterizing these interactions in cells presents a major challenge. Kleiner et al. describe a chemical proteomics approach combining photo-crosslinking and SILAC-based proteomics to characterize context-dependent direct protein-protein interactions in live cells. They apply their approach to profile interactions involving core histones H3 and H4.
KW - chemical proteomics
KW - chromatin
KW - protein-protein interactions
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U2 - 10.1016/j.chembiol.2017.10.001
DO - 10.1016/j.chembiol.2017.10.001
M3 - Article
C2 - 29104064
AN - SCOPUS:85034994098
SN - 2451-9456
VL - 25
SP - 110-120.e3
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 1
ER -