Zeeshan Hamid, Abdul Basit, Elisa Romeo and Andrea Armirotti
Istituto Italiano di Tecnologia, Italy
Scuola Superiore Sant�Anna, Italy
Posters & Accepted Abstracts: Biochem Mol biol J
Fatty acid amide hydrolase (FAAH) is a serine protease, which hydrolyzes bioactive endocannabinoids (ECs). ECs are lipid mediators that bind to and activate cannabinoid receptors CB1 and CB2. Upon binding ECs initiate a number of signaling pathways which control diverse set of biological functions including memory, learning, social behavior, appetite and inflammation. Modulating the metabolism of ECs by inhibiting FAAH holds therapeutic promise in a wide range of neurological diseases. Since the discovery of FAAH, it has been a very good target for pharmacological studies, many natural and synthetic analogues have been designed to target this enzyme. Given its large number of biological functions, a deeper understanding of its lipidome and proteome could generate useful information for future pharmacological strategies to combat neurological disorders. The aim of our work is to investigate the difference in lipidome of FAAH knockout mice brain compared to its wild type using ion mobility based mass spectrometry, difference in proteome using TMT labeled quantitative proteomics and a deeper targeted lipidomics of all the biosynthetic intermediates of anandamide pathway which is the most important signaling lipid in the brain. By using ion-mobility assisted, untargeted mass spectrometry, we discovered the upregulation of pro-apoptotic ceramides, besides previously known fatty acid ethanolamides and acyl taurines. Further we extended our study to TMT labeled quantitative proteomics to explore the effect of upregulated bioactive lipids on the proteome of FAAH knockout brain. The experiment generated relative expression data on various mouse proteins, out of which the majority of significantly upregulated proteins are related to cell growth, apoptosis, transport processes, cell to cell adhesion and RNA splicing. Our targeted lipidomic analysis of anandamide pathway showed a down-regulation of its metabolic precursors, which could be a possible feedback inhibition to fine tune the levels of anandamide. Overall our study gives a deeper understanding of the lipidome and proteome of FAAH knockout mice brain. In this seminar, I will discuss how to perform different omics techniques like untargeted lipidomics, untargeted proteomics and targeted lipidomics from a single experimental set of samples and how to predict different metabolic precursors of an unknown pathway and develop a reliable targeted LCMS method.
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