207 Tyrosine nitration induces a metabolic reprogram- ming in neurofibromatosis type 2-associated Schwannoma cells Jeanine Pestoni 1,* , Oliver Valdivia Camacho 1 , Ruben Riordan 1 , Sharon Kim 1 , Cristina Fernandez-Valle 2 , Maria Clara Franco 1 1 Oregon State University, USA 2 University of Central Florida, USA Neurofibromatosis type 2 (NF2) is a genetic tumor disorder of the ner- vous system caused by inactivation of the merlin tumor suppressor gene. The hallmark of the disease is the development of schwannomas along the acoustic nerve, and multiple other tumors for which there is no effective treatment. Production of peroxynitrite and subsequent tyrosine (Tyr) nitra- tion of proteins occurs in multiple tumor types. However, the role of Tyr nitration in tumorigenesis is unknown. We showed that schwannomas from NF2 patients and merlin-deficient (MD)-Schwann cells (NF2-associated schwannoma cell culture models) have significantly increased levels of Tyr nitration compared to wild-type (WT) Schwann cells. Further, prevention of tyrosine nitration significantly and selectively decreases MD-Schwann cell survival. Here we show that endogenous tyrosine nitration induces a me- tabolic reprogramming in human MD-Schwann cells characterized by: 1) decreased oxidative phosphorylation activity due to cytochrome coxidase inhibition, 2) increased glycolysis, and 3) almost complete glutamine de- pendency. Prevention of Tyr nitration reversed the metabolic phenotype of MD-Schwann cells back to that of WT-Schwann cells. We demonstrated that nitration of the chaperone Heat shock protein 90 (Hsp90) in tumor cells decreases mitochondrial metabolism. We show here that the intracellular delivery of nitrated Hsp90 in WT-Schwann cells significantly decreases mi- tochondrial oxygen consumption to levels comparable to those of MD- Schwann cells containing endogenous nitrated Hsp90. These observations suggest that nitrated Hsp90 participates in the metabolic reprogramming of MD-Schwann cells. Importantly, Hsp90 is not nitrated in any normal nervous tissue we have tested so far; implying that the nitrated form of Hsp90 could be an exceptional tumor-directed target for NF2 treatment in the short term. The identification of additional nitrated proteins that promote schwannoma metabolic reprogramming and growth could provide novel targets for the treatment of NF2 and potentially other tumor associated disorders. Sup- ported by DoD, NRP, W81XWH-17-1-0409 (to MCF). https://doi.org/10.1016/j.freeradbiomed.2018.10.212 208 Resurrecting an antibody against an alternative conformation of cytochrome c: the recombinant mAb 1D3 Rafael Radi 1,* , Florencia Tomasina 1 , Lucía Piacenza 1 , Natalie Castellana 2 , Matt Goff 3 , Tracy Cooper 3 , Hayes McDonald 3 , Stacey Seaback 3 , Paige Vinson 3 , Robert Carnahan 3 , Ronald Jemmerson 4 1 Universidad de la Republica, Uruguay 2 Digital Proteomics, USA 3 Vanderbilt University, USA 4 University of Minnesota, USA Cytochrome c can undergo reversible conformational changes under biologically-relevant conditions including oxidative post-translational modifications, interactions with phospholipids and electric fields. Revealing the existence and relevance of these alternative cytochrome c proteoforms at the cell and tissue level is challenging and has benefitted from im- munochemical approaches. Indeed, a monoclonal antibody that recognizes a major alternative conformation of cytochrome c, 1D3, was previously developed and characterized. The alternative conformation involves the destabilization of the Ω-loops encompassing residues 70 to 85 and 40 to 57 and the displacement of the Met80 heme ligand. However, the 1D3 pro- ducing hybridoma was lost and several attempts to reproduce it failed. Thus, with the remaining amounts of mAb 1D3 we aimed to “resurrect” 1D3 by the generation of a recombinant version of it. We employed the Valens algorithm for proteomic sequencing of 1D3. Briefly, the antibody was di- gested with multiple proteases and analyzed via LC-MS/MS using multiple fragmentation modes. By combining de novo peptide sequencing and homology mapping, we were able to obtain full-length sequences for the heavy and light chains of 1D3. Gene fragments representing the predicted heavy and light chain variable domains were synthesized using gBLOCK technology (IDT Inc., http://www.idtdna.com/). These were cloned into the heavy and light chain frameworks, respectively, using standard molecular biology techniques. Expression of light and heavy chains of 1D3 and its molecular assembly in HEK293 and CHO cells resulted in a functional an- tibody as tested by competitive ELISA with, for example, nitrotyrosine- containing cytochrome c species (NO274Tyr-Cyt c), known to adopt an al- ternative conformation. The recombinant 1D3 has the same specificity and affinity as the “original” 1D3 and is being successfully applied to various cellular models; enhanced levels of the 1D3-detectable alternative con- formation of cytochrome c and their nuclear translocation are observed under conditions of altered cell homeostasis. https://doi.org/10.1016/j.freeradbiomed.2018.10.213 209 Inhibition of respiratory chain Complex III irrever- sibly changes the mitochondria proteomic landscape Mikhail Vasilyev 1 , Cody Eccles 1 , Paul Langlais 1 , Olga Rafikova 1 , Stevan Tofovic 2 , Ruslan Rafikov 1,* 1 University of Arizona, USA 2 University of Pittsburgh, USA The role of mitochondria dysfunction in the pathogenesis of pulmonary hypertension (PH) is currently well-recognized. However, the particular mechanisms and the type of mitochondrial dysfunction are still being de- bated. We have recently shown that chronic inhibition of oxidative phos- phorylation by Antimycin A (AA) results in increased pulmonary pressure and remodeled pulmonary arteries. AA (0.35mg/kg) was given to rats three times during first six days and lungs were analyzed 30 minutes (acute ef- fect), 12 and 24 days (chronic effect) after the first AA injection. Mi- tochondria isolated from lungs were subjected to mass spectrometry and quantitative proteomic analysis to estimate changes in the isolated mi- tochondrial proteome over the course of disease development. Using a 4-fold change cutoff value, 48 mitochondrial proteins were discovered to be altered upon AA treatment, with 13 proteins exhibiting a significant dif- ference. Our data indicate that proteomic data with applied 4-fold cutoff can completely distinguish between the control and 24-day groups using principal component analysis. Functional analysis of the proteome data revealed major downregulation of enzymes involved in fatty acids oxida- tion(ACDSB,HADH) and fatty acids transport(CPT1, ACSM5). Electron transport chain proteins showed mixed results with downregulation of Complex IV (COX1/2), altered Complex I subunits(NU4/5M) and assembly protein expression(NDUF4), and upregulation of ubiquinone biosynthesis (COQ3/7). Activation of ubiquinone biosynthesis could be explained as compensation for inhibition of mitochondrial respiration by AA. Mi- tochondrial machinery for importing proteins into the matrix(TIM9/10/13) was upregulated, but peptidases(LON,MPPA) that control the quality of A. Newman / Free Radical Biology and Medicine 128 (2018) S79–S97 S91