We are interested in studying the biological mechanisms of lipid signaling pathways in the central nervous and immune systems. To achieve our goals, we integrate aspects of chemical biology, immunology, animal, and/or cellular models, in conjunction with advanced mass spectrometry-based metabolomics (lipidomics) and (chemo)proteomics techniques. Our long term goal is to identify and characterize as-of-yet uncharacterized lipid signaling pathways in vivo, annotate enzymes and/or cognate receptors that regulate their biology, and provide new insights and therapeutic paradigms for orphan and/or emerging human diseases.

UNDERSTANDING THE MOLECULAR BASIS OF PHARC

Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is an autosomal recessive early onset neurological disorder in humans caused by deleterious mutations to the abhd12 gene that encodes a metabolic serine hydrolase (lipase) enzyme ABHD12. It has been recently shown that the enzyme functions as a major lysophosphatidylserine (lyso-PS) lipase in the mammalian central nervous and immune system (Figure 1), where it controls the levels of immunomodulatory lyso-PS lipids. While a lot is known of the immune phenotype regulated by lyso-PS lipids, their receptors and/or protein ligands remain unknown, and we are interested in finding their identities and characterizing them in lyso-PS mediated immunological processes. Additionally, the integral membrane enzyme ABHD16A, is an upstream phosphatidylserine (PS) lipase that makes lyso-PS in the mammalian central nervous and immune systems (Figure 1), and we are interested in understanding the role of this lipase in the context of PHARC, and hereditary spastic paraplegia that it has now been associated with.

Selected references:

1. Khandelwal, N. et al., Cell Chemical Biology (2021)

2. Singh, S. et al., European Journal of Neuroscience (2021)

3. Singh, S. et al., Biochemistry (2020)

4. Joshi, A. et al., Journal of Biological Chemistry (2018)

Figure 1. A schematic of the ABHD12/ABHD16A-lyso-PS pathway in mammals

MAPPING LIPID PATHWAYS DURING OXIDATIVE STRESS

Lipids are the primary targets of oxidative damage for cells that have elevated ROS levels because of oxidative stress. Several oxidatively damaged lipids serve as key immunological modulators, yet their chemical structures, their metabolic pathways and receptors remains unknown. We have recently elucidated the chemical structures of the pro-apoptotic oxidized phosphatidylserine (ox-PS) lipids, and shown that ABHD12 is a major lipase that controls their concentrations in vivo under chronic oxidative stress (Figure 2). Moving forward, we are now interested in identifying other lipid classes (e.g. sterols, neutral lipids) that might be susceptible to oxidative damage using the same strategy to understand their chemical structures and the metabolic and/or signalling pathways that they perturb during oxidative stress in mammalian cells and tissues.

Selected references:

1. Kelkar, D. et al., Nature Chemical Biology (2019)

2. Chaplot, K. et al., Disease Models & Mechanisms (2019)

Figure 2. A schematic representation of the oxidised PS and lyso-PS lipase activities of ABHD12, and the contribution of these signalling lipids to the pathology of PHARC.

The ROS generating probe MGR1 and the corresponding inactive control compound MGR2 generated during this project are now available from Merck (Sigma-Aldrich)

CHARACTERIZATION OF THE ABHD14 ENZYMES

We have recently annotated the orphan metabolic serine hydrolase enzyme ABHD14B as a novel lysine deacetylase (Figure 3), and shown it to catalyses a unique deacetylase (hydrolytic) reaction. Interestingly, ABHD14B has very restricted expression in the metabolically active tissues (liver & kidneys) in mammals, and we are interested in investigating the biological pathways under the control of ABHD14B, and its role as a regulator of central metabolism in mammals.

Selected reference:

1. Rajendran, A. et al., Journal of Biological Chemistry (2022)

2. Rajendran, A. et al., Biochemistry (2020)

Figure 3. The lysine deacetylase activity of the orphan metabolic serine hydrolase ABHD14B.

MAPPING LIPID PATHWAYS DURING PHAGOCYTOSIS

Phagocytosis is an evolutionary conserved process, involving interplay between various cellular organelles, and their membranes. We recently performed a rigorous lipidomics study of phagosomes of various ages, and showed for the first time, that the sphingolipids are critical for phagosomal maturation (Figure 4). We have also mapped changes in phospholipid pools during phagocytosis, and are also interested in annotating function to putative phospholipases involved in process.

Selected references:

1. Saharan, O. et al., Biochem. Soc. Trans. (2023)

2. Saharan, O. et al., Curr. Res. Chem. Biol. (2022)

3. Mehendale, N. et al., ACS Chemical Biology (2021)

4. Pathak, D. et al., ACS Chemical Biology (2018)

Figure 4. A summary of the sphingolipid pathways mapped by our lab during phagosomal maturation.

OUR LIPIDOMICS & CHEMOPROTEOMICS PLATFORMS

Since liquid chromatography coupled to mass spectrometry (LC-MS/MS) based lipidomics & (chemo)proteomics are central to all the ongoing projects in the lab, we have set up a Biological Mass Spectrometry lab at the Department of Biology at IISER Pune that specializes in: (i) quantitative lipidomics platform for profiling lipids (Figure 5), and (ii) discovery and quantitative chemoproteomics platform for profiling protein activities (Figure 6). Several collaborative projects have emerged from the use of these platforms.

Our lipid (or metabolite) extractions and LC-MS/MS platform now allows for the detection and quantitative assessment of >2000 unique lipid species, including phospholipids and their lyso-versions, neutral lipids, sterols and their esters, sphingolipids, and oxidatively truncated and/or damaged lipids of aforementioned classes, while our chemoproteomics platforms, termed activity based protein profiling (ABPP) (pioneered by Ben Cravatt's lab) allow for discovery data dependent and independent chemoproteomics, and assessing endogenous enzyme activities in complex biological settings.

Selected recent references for the lipidomics platforms:

1. Mondal, S. et al., eLife (2022)

2. Kumar, S. et al., mBio (2022)

3. Mehdiratta, K. et al., PNAS (2022)

Selected recent references for the chemoproteomics platforms:

1. Kumari, P. et al. Microbiology Spectrum (2023)

2. Shanbhag, K. et al., RSC Chemical Biology (2023)

3. Kumar, K. et al., Biochemistry (2021)

Figure 5: Schematic of a pipeline to quantitatively measure lipids (and other metabolites) from complex biological samples (e.g. cells, tissues).

Figure 6. The activity based protein profiling (ABPP) technology pioneered by Ben Cravatt's lab. There are several platforms to which the ABPP technology can be applied; shown above is the gel-based ABPP platform, and the LC-MS/MS based multidimensional protein identification technology (ABPP-MudPIT) platform.