Many phospholipids in the cell membrane play important regulatory roles, and these signaling lipids interact with proteins to effect the regulation. Our interest in this area started with the discovery of phosphatidic acid regulation of mTOR signaling many year ago, and has recently expanded to phosphoinositide signaling. Revealing the specificity of lipid-protein interactions is pivotal to the understanding of lipid signaling, and a critical need is experimental approaches that enable investigation of such interactions in physiologically relevant contexts. In collaboration with Taekjip Ha Laboratory we developed a single-molecule pulldown (SiMPull) assay to study the interaction between lipid vesicles (or nanodiscs) and proteins of interest in crude cell lysates with high sensitivity and specificity. This assay allowed us to re-evaluate phosphoinositide binding by PH domain-containing proteins, the largest family of putative lipid binding proteins in the human proteome, which led to the discovery of lipid binding specificity not previously appreciated. Armed with this powerful approach, we are investigating lipid signaling to gain novel biological insights.
A. Jain, R. Liu, B. Ramani, E. Arauz, Y. Ishitsuka, K. Ragunathan, J. Park, J. Chen, Y. K. Xiang, T. Ha, (2011) Probing cellular protein complexes using single-molecule pull-down, Nature 473, 484-488. [News & Views, Nature 473, 461-462] Link to article
A. Jain, E. Arauz, V. Aggarwal, J. Chen*, and T. Ha*, (2014) Stoichiometry and Assembly of mTOR Complexes Revealed by Single-Molecule Pull-Down. Proc. Natl. Acad. Sci. USA, 111, 17833-8. (*Co-corresponding authors) Link to article
E. Arauz, V. Aggarwal, A. Jain, T. Ha, and J. Chen, (2016) Single-Molecule Analysis of Lipid-Protein Interactions in Crude Cell Lysates, Anal. Chem., 88, 4269-76. Link to article
N. Singh, A. Reyes-Ordonez, M.A. Compagnone, J.F. Moreno, B.J. Leslie, T. Ha, and J. Chen, (2021) Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins. Nat. Commun, 12, 4339. Link to article
J. F. Moreno, J.-S. You, C. C. Rodriguez, S. Pant, A. Reyes-Ordoñez, R. Abdullah, N. Singh, M. J. van der Donk, E. Tajkhorshid, and J. Chen, (2025) Regulation of Rho guanine nucleotide exchange factor 3 (ARHGEF3) by phosphorylation in the PH domain. iScience, 28(6):112753. Link to article
Reyes-Ordoñez A, Shree S, Singh N, Sligar SG, Chen J, (2025) Nanodisc single-molecule pulldown to study lipid-protein interactions. J Lipid Res. 66(7):100846. Link to article