Motif-dependent immune co-receptor interactome profiling by photoaffinity chemical proteomics

Xiong Chen; Shanping Ji; Zheyi Liu; Xiao Yuan; Congsheng Xu; Ruxi Qi; An He; Heng Zhao; Haiping Song; Chunlei Xiao; Weina Gao; Peng R Chen; Ray Luo; Pengfei Li; Fangjun Wang; Xueming Yang; Ruijun Tian

doi:10.1016/j.chembiol.2022.01.005

Motif-dependent immune co-receptor interactome profiling by photoaffinity chemical proteomics

Cell Chem Biol. 2022 Jun 16;29(6):1024-1036.e5. doi: 10.1016/j.chembiol.2022.01.005. Epub 2022 Jan 31.

Authors

Xiong Chen¹, Shanping Ji¹, Zheyi Liu², Xiao Yuan¹, Congsheng Xu¹, Ruxi Qi³, An He¹, Heng Zhao⁴, Haiping Song⁵, Chunlei Xiao⁶, Weina Gao¹, Peng R Chen⁵, Ray Luo¹, Pengfei Li⁷, Fangjun Wang⁸, Xueming Yang⁹, Ruijun Tian¹⁰

Affiliations

¹ Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China.
² CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
³ Cryo-EM Center, Southern University of Science and Technology, Shenzhen 518055, China.
⁴ CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
⁵ Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
⁶ State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
⁷ Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: lipf@sustech.edu.cn.
⁸ CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: wangfj@dicp.ac.cn.
⁹ Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China; State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
¹⁰ Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China; Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: tianrj@sustech.edu.cn.

PMID: 35093210
DOI: 10.1016/j.chembiol.2022.01.005

Abstract

Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28^cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28^cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28^cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.

Keywords: C2 domain; CD28; PKCθ; chemical proteomics; immune signaling; phosphotyrosine signaling; protein complex.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CD28 Antigens* / chemistry
CD28 Antigens* / metabolism
Mass Spectrometry
Phosphorylation
Proteomics*
Signal Transduction

Substances

CD28 Antigens