Schopfer Lab

Tha Schopfer Lab studies the role of bioactive fatty acids in physiology and pathology. Using cutting edge mass spectrometry-based lipidomic approaches we identified novel signaling lipids in vivo. Using cellular and animal models we investigate the biological and biochemical activity of these compounds. Among the fatty acids we discovered, we focus our investigation on nitrated and furan fatty acids. Nitrated fatty acids are potent anti-inflammatory and anti-oxidant endogenous fatty acids that we validated in preclinical animal models successfully moved to Phase I and II clinical trials. Furan fatty acids are an important and neglected component of fish oils and we investigate their signaling activities. Overall, to further support our early discovery efforts and pharmacological developments, we use organic synthesis, isotopically labeled standards, pharmacokinetic evaluations, formulation development and pharmacodynamics approaches. 

Below displays the Research Details from the profile of each member of the lab.

Pascal Rowart, PhD

Metabolic syndrome is a multifactorial disease that affects more than 35% of the world’s population and is associated with comorbidities including heart disease and cardiovascular dysfunction thus reducing life expectancy and quality. Metabolic syndrome is characterized by increased plasma levels of cholesterol, triglycerides (TG), insulin resistance, hypertension, an increased inflammatory state, and risk of cardiovascular diseases (CVD) events. Fish oil (FO) derived omega-3 (ω-3) have been shown to be protective and are used in the clinic to reduce circulating triglycerides, reduce the risk of adverse cardiovascular events and plaque formation.
 
To characterize potential bioactive metabolites of ω-3 treatments involved in these beneficial effects, We assessed samples from two clinical trials involving healthy human volunteers receiving the ω-3 FA prescription drug Lovaza using untargeted metabolomic approaches. To our surprise, we found elevated levels of circulating and urinary metabolites (CMPF) of furan FA (FuFA). FuFA are a ubiquitous class of FA mainly found in FO, making up to 3% of total FA. 



I study the effect of FuFA on metabolic syndrome, obesity, non-alcoholic fatty liver disease and diabetes. I am using new drug based on fish oil and omega-3 fatty acids to prevent metabolic syndrome, obesity-related cardiovascular disease, and hypertriglyceridemia.
 
Finally, I am working on another project related with nitrated fatty acids (NO2-FA) which are electrophilic molecules present in plants, animals, and humans that form during digestion and inflammation and display pleiotropic signaling activities. A unique pharmacokinetic profile is expected for NO2-FA because of an ability to undergo reversible reactions including Michael addition with cysteine-containing proteins and esterification into complex lipids. The signaling actions of NO2-FA have been extensively studied, but their detection and characterization lagged. We are aiming at understanding their mode of action using in vivo and in vitro models.
 

Francisco J. Schopfer, PhD

Dr. Schopfer’s research is focused on the understanding of the biological effects of electrophilic fatty acids. In particular, he studies the mechanism by which nitrated fatty acid activate and signal through peroxisome proliferator-activated receptor gamma (PPARγ).  This receptor is the target of currently used antidiabetic drugs (thiazolidinediones). The activation of the receptor regulates fat and glucose metabolism, resulting in an overall decrease of glucose levels to normal values in patients with type II diabetes. The targeting of this receptor by nitrated fatty acids results in a decrease of the glucose levels to normal values like thiazolidinediones, but without the known secondary effects exerted by thiazolidinediones. In addition to the intrinsic therapeutic value of nitrated fatty acid, they will aid in the understanding of the biological mechanism involved in PPARγ activation, leading to improved designs of anti-diabetic drugs targeting the PPARγ receptor.

Figure 1

Fig 1. Modeling of PPARγ receptor activation by nitrated oleic acid

Biological effects of endogenous PPARϒ ligands

The role of the PPARγ receptor in diabetes has been well established. Nonetheless, the role of endogenous signaling molecules on the activation of PPARϒ is still unclear and under debate. Nitrated fatty acids are endogenously formed and bind to PPARγ with high affinity rivaling Rosiglitazone (thiazolidinediones), resulting in receptor activation. In addition, nitrated fatty acids covalently modify a critical cysteine (cys285) in the ligand binding pocket of PPARγ promoting a particular conformational change that results in partial receptor activation. This partial activation results in the expression of a particular subset of genes under PPARγ regulation and a biological outcome that differs from the one obtained when activating the receptor with Rosiglitazone. Dr. Schopfer’s work focuses on understanding the mechanism of this selective activation and how it avoids the side effect presented upon full activation by agonist like Rosiglitazone.
 
Endogenous formation of electrophiles and biological relevance
 
Electrophilic fatty acids are constantly formed as fatty acid breakdown products during oxidative stress and as signaling messengers by enzymatic or non enzymatic pathways. Dr. Schopfer studies the formation of biologically relevant electrophiles, in particular nitrated fatty acids, and their signaling mechanisms. The study involves the detection and characterization of novel electrophiles formed during inflammation. Once the molecules are characterized, a chemical synthesis approach is used to generate enough quantities for biological experiments.

Figure 2

Fig 2 A Key Reactivity of nitrated fatty acids: rapid, reversible Michael Addition reactions.

Proteomic analysis of electrophilic post-translational modifications

Electrophiles induce an important cellular response that includes the induction of phase II genes. This will in turn set up a more protective environment against damaging electrophilic molecules. A key player in the initiation of this biological response is the Keap 1/Nrf 2 couple. Keap 1 is usually bound to Nrf 2 in the cytoplasm. Upon formation of electrophiles, Keap 1, which contains several highly reactive cysteine, is targeted, dissociates from Nrf2 and is routed to degradation by the proteosome. These lead to Nrf2 nuclear translocation and activation of phase II genes. In particular, we study the mechanism by which different biologically relevant electrophiles target KEAP 1 and activate Nrf 2 responses. In addition, a more general proteomic approach is use to evaluate and characterize different electrophilic cellular protein targets. Once critical targets are identified using a mass spectrometry approach, a functional study of the modification is performed to determine the relevance and its cellular effects.


Pascal Rowart, PhD
Research Instructor


Sonia Salvatore
Research III


Francisco J. Schopfer, PhD
Associate Professor & Vice Chair for Biotechnology Development

Pascal Rowart, PhD

Journal Articles

Jobbagy S, Vitturi DA, Salvatore SR, Pires M, Rowart P, Emlet DR, Ross M, Hahn S, St. Croix C, Wendell SG, Subramanya AR, Straub AC, Tan RJ and Schopfer FJ. Nrf2 activation protects against lithium-induced nephrogenic diabetes insipidus. Journal of Clinical Investigation Insight 5(1):e128578, 2020.
Salvatore SR, Rowart P and Schopfer FJ. Mass spectrometry-based study defines the human urine nitrolipidome. Free Radical Biology and Medicine 162:327-337, 2020.
Wu J, Rowart P,  Gassaway BM, Rinehart J, Rajendran V, Jouret F and Caplan MJ. Mechanisms Iinvolved in AMPK-mediated deposition of tight junction components to the plasma membrane. American Journal of Physiology, Cell physiology 10:1152/ajpcell.00422.2019, 2019.
Rowart P, Wu J, Caplan MJ and Jouret F. Implications of AMPK in the formation of epithelial tight junctions. International Journal of Molecular Sciences 19(7):2040, 2018.
Rowart P, Erpicum P, Krzesinski JM, Sebbagh M and Jouret F. Mesenchymal stromal cells accelerate epithelial tight junction Assembly via the AMP-activated protein kinase pathway, independently of liver kinase B1. Stem Cells International, Volume 2017, 2017. Article ID 9717353
Erpicum P, Rowart P, Poma L, Krzesinski JM, Detry O and Jouret F. Administration of mesenchymal stromal cells before renal ischemia/reperfusion attenuates kidney injury and may modulate renal lipid metabolism in rats. Scientific Report 7:8687, 2017.
Rowart P, Erpicum P, Detry O, Weekers L, Grégoire C, Lechanteur C, Briquet A, Beguin Y, Krzesinski JM and Jouret F. Mesenchymal stromal cell therapy in ischemia/reperfusion injury. Journal of Immunology Research, Volume 2015, 2015. Article ID 602597
Fazzari M, Woodcock SR, Rowart P, Ricart K, Lancaster JR, Patel R, Vitturi DA, Freeman BA and Schopfer FJ. Endogenous generation of nitro-fatty acid hybrids having dual nitrate ester (RONO2) and nitroalkene (RNO2) substituents. Redox Biology 41:101913, 2021.

Francisco J. Schopfer, PhD

Journal Articles

Jobbagy S, DA Vitturi, SR Salvatore, L Turell, MF Pires, E Kansanen, C Batthyany, JR Lancaster, Jr., BA Freeman and FJ Schopfer.  Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis. Redox Biol 21, 101050, 2019.
Fazzari M, DA Vitturi, SR Woodcock, SR Salvatore, BA Freeman and FJ Schopfer. Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids. J Lipid Res 60:388-399, 2019.
Hansen AL, GJ Buchan, M Ruhl, K Mukai, SR Salvatore, E Ogawa, SD Andersen, MB Iversen, AL Thielke, C Gunderstofte, M Motwani, CT Moller, AS Jakobsen, KA Fitzgerald, J Roos, R Lin, TJ Maier, R Goldbach-Mansky, CA Miner, W Qian, JJ Miner, RE Rigby, J Rehwinkel, MR Jakobsen, H Arai, Y Taguchi, FJ Schopfer, D Olagnier and CK Holm. Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling. Proc Natl Acad Sci U S A 115:E7768-E7775, 2018,
Prentice KJ, SG Wendell, Y Liu, JA Eversley, SR Salvatore, H Mohan, SL Brandt, AC Adams, X Serena Wang, D Wei, GA FitzGerald, TB Durham, CD Hammond, KW Sloop, C Skarke, FJ Schopfer and MB Wheeler.  CMPF, a metabolite formed upon prescription omega-3-acid ethyl ester supplementation, prevents and reverses steatosis. EBioMedicine 27:200-213, 2018,
Turell L, DA Vitturi, EL Coitino, L Lebrato, MN Moller, C Sagasti, SR Salvatore, SR Woodcock, B Alvarez and FJ Schopfer. The chemical basis of thiol addition to nitro-conjugated linoleic acid, a protective cell-signaling lipid. J Biol Chem 292:1145-1159, 2017.
Fazzari M, N Khoo, SR Woodcock, L Li, BA Freeman and FJ Schopfer.Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides. Free Radic Biol Med 87:113-124, 2015.
Vitturi DA, L Minarrieta, SR Salvatore, EM Postlethwait, M Fazzari, G Ferrer-Sueta, JR Lancaster, Jr., BA Freeman and FJ Schopfer. Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride. Nat Chem Biol 11:504-510, 2015.
Vitturi DA, CS Chen, SR Woodcock, SR Salvatore, G Bonacci, JR Koenitzer, NA Stewart, N Wakabayashi, TW Kensler, BA Freeman and FJ Schopfer. Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase. J Biol Chem 288:25626-25637, 2013.
Bonacci G, PR Baker, SR Salvatore, D Shores, NK Khoo, JR Koenitzer, DA Vitturi, SR Woodcock, F Golin-Bisello, MP Cole, S Watkins, C St Croix, CI Batthyany, BA Freeman and FJ Schopfer. Conjugated linoleic acid is a preferential substrate for fatty acid nitration. J Biol Chem 287:44071-44082, 2012.
Groeger AL, C Cipollina, MP Cole, SR Woodcock, G Bonacci, TK Rudolph, V Rudolph, BA Freeman and FJ Schopfer.  Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids.  Nat Chem Biol 6:433-441, 2010.

Reviews

Hansen AL, K Mukai, FJ Schopfer, T Taguchi and CK Holm. STING palmitoylation as a therapeutic target. Cell Mol Immunol 16:236-241, 2019.
Schopfer FJ and NKH Khoo. Nitro-fatty acid logistics: Formation, biodistribution, signaling, and pharmacology. Trends Endocrinol Metab 30:505-519, 2019.
Schopfer FJ, DA Vitturi, DK Jorkasky and BA Freeman. Nitro-fatty acids: New drug candidates for chronic inflammatory and fibrotic diseases. Nitric Oxide 79:31-37, 2018,
Freeman BA, VB O'Donnell and FJ Schopfer. The discovery of nitro-fatty acids as products of metabolic and inflammatory reactions and mediators of adaptive cell signaling. Nitric Oxide 77:106-111, 2018.
Schopfer FJ, C Cipollina and BA Freeman. Formation and signaling actions of electrophilic lipids. Chem Rev 111:5997-6021, 2011.