Lightning Talk & Poster 27th Annual Lorne Proteomics Symposium 2022

Mapping cysteine exposure of proteins in situ with a bi-functional aggregation-induced emission fluorescent probe (#18)

Shouxiang Zhang 1 , Tze Cin Owyong 2 , Paul Fisher 3 , Yuning Hong 1
  1. Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia
  2. ARC Centre of Excellence in Exciton Science, School of Chemistry, Bio21 Institute, The University of Melbourne, Melbourne, VIC, Australia
  3. Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, VIC, Australia

Proteostasis networks maintain proteins in a defined conformation for their activity, localization, function and interaction. Endogenous or exogenous stressors can perturb proteostasis integrity and deplete folding capacity, generating destabilized unfolded proteins and protein aggregates. Mechanisms of how proteostasis network and protein quality control interact and coordinate with each other are yet to be fully understood. Here, we report an aggregation-induced emission fluorescent probe, TPE-EMI, for quantitation of unfolded proteins in cells by both fluorescence measurement and proteomics approach in a high-throughput way. TPE-EMI is inherently non-fluorescent but becomes fluorescent upon reacting with free cysteine thiols in unfolded proteins.1 Results show that the reactivity of TPE-EMI towards free thiols depends on both the surface exposure and nearby hydrophobic environment, rendering its specificity for unfolded proteins. Based on a two-state folding model, TPE-EMI is capable to profile protein denaturation curve and quantitate protein stability in terms of thermodynamic measurement. With a latent analytical tag, TPE-EMI is shown to enrich and identify unfolded proteins in cells using affinity purification-mass spectrometry. Functional enrichment analysis2 and protein unfoldedness prediction3 reveal that these TPE-EMI binders are enriched in intrinsically disordered proteins (IDPs) that play an essential role in biological processes and protein-protein interaction networks. TPE-EMI is further applied in a Huntington’s disease model and pulse-shape analysis (PulSA)4 show that aggregation-prone proteins undergo a characteristic 3-stage pattern, and protein aggregation is protective against proteostasis imbalance. To rejuvenate cell folding capacity, cells tend to sequester not only unfolded and misfolded but also overexpressed proteins into locally aggresome-like structures or bulky inclusions as a generic strategy for cellular quality control. Collectively, TPE-EMI makes a novel tool for mechanistic research of protein unfolding and aggregation, investigation of stress response, discovery of novel biomarkers and drug targets, as well as potential clinical applications, e.g., disease diagnosis.

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  2. T. Wu, E. Hu, S. Xu, M. Chen, P. Guo, Z. Dai, T. Feng, L. Zhou, W. Tang, L. Zhan, X. Fu, S. Liu, X. Bo and G. Yu, clusterProfiler 4.0: A universal enrichment tool for interpreting omics data, The Innovation, 2021, 2, 100141.
  3. B. Mészáros, G. Erdős and Z. Dosztányi, IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding, Nucleic Acids Research, 2018, 46, W329-W337.
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