Research Summary: Signalling lipid–protein interactions influence several processes involved in cellular homeostasis and signalling. We developed the photo-affinity labelling-based chemical proteomics to map the protein interactome of a particular class of lipid called monoacylglycerols (MAGs).
First author: Dr Karthik Shanbhag was a PhD student at Dr. Siddhesh Kamat’s lab at IISER Pune. There he developed the chemical proteomic screening strategy for mapping the protein interactome of signalling lipids. Currently, he is a senior research scientist at Ahammune Biosciences Pvt Ltd.
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Lab: Dr Siddhesh S Kamat, IISER Pune
Author interview
What was the core problem you aimed to solve with this research?
Signalling lipids play a crucial role in modulating various biological processes, and any dysregulation/imbalance of such lipids could lead to various neurological, metabolic, and autoimmune diseases. The biological processes modulated by these lipids occur via the interaction of these lipids with various proteins, such as enzymes, receptors, transporters, etc., which in turn activate/repress signalling pathways within the cells.
Our aim was to decipher the protein interactome of one such lipid class called monoacylglycerols (MAGs). Among this lipid class, only 2-arachidonoylglycerol (2-AG) has been well studied owing to being an endocannabinoid, but the same cannot be said about the other species belonging to the MAG lipid class. Therefore, mapping an interactome of MAGs would shed light on the signalling pathways modulated by MAGs.
How did you go about solving this problem?
In recent years, there have been groundbreaking advances in the field of photoaffinity labelling (PAL). This technique allows us to map the entire proteome of any metabolite in question, which in our case was a lipid, more specifically, MAGs. To accomplish this, our lab synthesized a bifunctional lipid probe of MAGs. The lipid probe is a naturally occurring lipid that has been appended to two additional functional groups (hence, it is called bifunctional). Using these lipid probes, we were able to delineate the protein interactome of MAGs in mouse brain tissue and various other cell lines via mass spectrometry-based chemical proteomics. After screening through the protein interactors of MAGs, we selected a protein called Hippocalcin (HPCA) for further molecular docking and biochemical studies. The molecular docking studies showed that HPCA has a hydrophobic pocket that can bind to MAGs, which served as proof that our proteomic screen successfully identified protein interactors of MAGs. Further, our biochemical studies with the purified HPCA protein revealed that HPCA was indeed an interactor of MAGs and that MAGs modulate the calcium sensing capability of HPCA in neurons. And we further reported that interaction of MAGs with HPCA influences the binding of HPCA to AP2 adaptor protein in neurons, which in turn modulates the internalization/endocytosis of AMPA receptors. This finding revealed that MAGs may play a significant role in calcium signalling in neurons and may also affect the synaptic activity in neurons.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Lipids (or, in general terms, fats) are one of the crucial biomolecules in cells. In recent years, lipids have been found to be involved in cellular signalling (communication between cells). An imbalance in these lipids is known to cause metabolic disorders (such as Niemann-Pick disease, Gaucher’s disease, etc.) and neurological disorders (such as PHARC, hereditary Spastic Paraplegia, etc.). Therefore, understanding the roles these lipids play in cells can help us in designing new drugs and therapeutic interventions to treat diseases.
The first step towards understanding how these lipids affect cells is to find the proteins they interact with, which can help us better understand the signalling cascade they activate.
The PAL-based chemoproteomics platform allows us to fish out the protein interactors of these signalling lipids. And as mentioned earlier, by finding these protein interactors, we can design new drugs and treatment approaches to combat the diseases caused due to imbalance of these lipids.
“Photoaffinity labelling in conjunction with chemoproteomics is emerging as an excellent technology to identify hitherto unknown protein ligands to signalling lipids. In this exciting study, leveraging this principle, we provide a resource for the community to study new protein functions in the context of MAG lipids”
What are the potential implications of your findings for the field and society?
Using the PAL-based chemoproteomic platform, we mapped the protein interactome of a lipid class called MAGs. This enabled us to find a putative interactor of MAG, a protein called Hippocalcin (HPCA). HPCA is a calcium-sensing protein in neurons, one of the crucial proteins involved in neuronal calcium signalling and regulates synaptic activity in neurons. Therefore, we believe that MAGs may serve as a potential link in understanding this particular pathway. Furthermore, mutations in the HPCA protein are known to cause a rare autosomal recessive neurological disorder called Dystonia in humans. Therefore, we firmly believe that the discovery of the interaction between HPCA and MAG lipids may potentially lead us to new medical avenues in treating this disorder.
Additionally, our research provides a blueprint for mapping the protein interactome of any biomolecules, be it lipids, carbohydrates, vitamins, cofactors, etc. This will further our understanding of the roles of these biomolecules in cellular signalling, which could, in turn, lead us to novel therapies/medicines for treating various symptoms and disorders associated with these biomolecules.
What was the exciting moment during your research?
Our Study had two main objectives; First was to standardize the PAL-based chemoproteomic screening strategy for the in-house synthesized MAG lipid probe. The second was to select the putative protein interactors from several hundred proteins we obtained from the proteomic screen. Accomplishing both these aims was exciting in their own ways.
Initially, we standardized the usage of these probes using an in-gel fluorescence-based method in the mouse brain and various cell lines. Once the technique was standardized, we had to perform mass spectrometry-based chemoproteomic screening for identifying proteins that may interact with MAGs. And this was one of the most time-consuming and daunting tasks to accomplish, since we obtained over thousands of proteins through our proteomic screen. Combining through each and every data set obtained from the proteomic screen to shortlist a few dozen proteins that may be putative interactors of MAGs was challenging, since we had no prior knowledge about the protein interactors of MAGs. Selecting a wrong protein would mean we would have to start all over again to find another protein that may interact with MAGs.
After an extensive literature review and rigorous screening, we chose HPCA for our successive experiments. For a scientist, there is no greater pleasure than obtaining results that prove their hypothesis. Since there were no studies/literature that mentioned HPCA as an interactor of MAGs, it was actually one of the most thrilling discoveries, that it indeed was an interactor of MAG lipids. Further, building up on our hypothesis, we reported that MAGs do, in fact, influence the calcium sensing property of HPCA and its ability to bind to AP2 adaptor protein in neurons. Interaction between HPCA and AP2 in neurons is known to influence the internalization of AMPA receptors in neurons, affecting the synaptic activity in neurons. Although more studies are required to understand the entire signalling pathway, our studies show that MAGs may have a significant role in influencing neuronal signalling. We sincerely hope our research encourages fellow scientists to look into this pathway more closely and delineate it.
Paper reference: Shanbhag, K., Mhetre, A.B., Saharan, O. et al. Chemoproteomics identifies protein ligands for monoacylglycerol lipids. Commun Chem 8, 197 (2025). https://doi.org/10.1038/s42004-025-01589-w
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