Periodic Reporting for period 2 - CellCellEM (Communications at the Synapse - a Near Atomic Resolution View into Cell-Cell Communication in the CNS)
Reporting period: 2022-05-01 to 2023-10-31
The CellCellEM project aims at understanding cellular communication in the brain at the molecular level through structural studies of receptors in the central nervous system (CNS). The project focuses on adhesion G protein coupled receptors (aGPCRs), a class of receptors that are highly abundant in the CNS, aiming to uncover their enigmatic mechanism of action through comprehensive analysis of their structure and function. The project also encompasses a broader exploration of cellular communication in the brain, focusing on investigating protein-protein interactions at the cellular interface. The overarching objective involves the characterization of both the structural and functional aspects, including signaling, of proteins residing within the CNS, as well as their intracellular and extracellular interactome. The principal method employed for this investigation is single particle cryo-electron microscopy (cryo-EM), a technique enabling the capture of detailed snapshots of macromolecules at distinct conformational states and at high resolutions. This project addresses a long-standing question regarding the operational mechanisms of CNS receptors, and its outcomes are anticipated to contribute to future endeavors aimed at targeting these proteins, which are also implicated in CNS disorders.
Achievements in the project included the determination of high-resolution structures of adhesion GPCRs (aGPCRs) captured in active and inactive states (Barros-Álvarez et. al. Nature 2022). The intracellular signaling pathways of these receptors were also delineated through this work, and a structure of receptor bound to its intracellular partners was published in collaboration with two groups at Stanford and at the University of Michigan. Collectively, these findings unveiled the distinctive signaling mechanism employed by this particular class of receptors within the brain, and paved the way for structure based drug design. Present efforts center on the investigation of aGPCR interactions with extracellular ligands, further expanding our knowledge regarding their CNS signaling.
The main goal of CellCellEM is to delineate cellular communication through understanding signaling at the structural and at the molecular levels. The project was designed to primarily characterize the structure and function of individual signaling proteins residing at the cellular membrane, but also to understand the way they communicate with their partners within the cell, outside the cell, and inside the cellular membrane. Specifically, the project focused on a family of adhesion GPCRs, the latrophilins, that are highly abundant in the CNS and are known to maintain multifaced interactions with proteins within the cell, the cellular membrane and in neighboring cells. However, while the initial scope of the project was to focus on this family of proteins, the project expanded way beyond the current scope, both through working on additional CNS proteins (e.g. other GPCRs) and also for the exploration of the evolutionary evolvement of signaling. These two avenues culminated in an ongoing work in our group that lead to the discovery of a novel family of proteins, the bestrhodopsins, that are light activated ion channels composed of rhodopsins fused to bestrophin ion channels, and at the determination of additional GPCR structures of other components of the CNS. A paper describing the bestrhodopsin discovery was recently published by our group (Rozenberg et al. Nature Struct. Mol. Biol. 2022), while the later studies on GPCRs are currently in final preparations of manuscripts to be sent within the next few weeks. The study on bestrhodopsins is currently ongoing in the lab and as rhodopsins are considered to be ancestors of GPCRs, these studies may potentially provide hints regarding the evolution of signaling mechanisms and aid in understanding the inner workings of the more complex signaling pathways in our brain. Altogether these studies are an evolvement of the suggested ERC project and are conducted in parallel to our work on the explicit aims 2 and 3 mentioned in the proposal, that are aimed at the structural and functional studies of latrophilins with their extracellular partners.