Elizabeth A. Sweeny, PhD
Heme proteins play essential roles in several critical cellular processes, including gas exchange, catalysis, electron transfer reactions, transcriptional control, and initiation and propagation of signaling cascades. Consequently, dysfunction of heme proteins contributes to many human diseases including pulmonary and cardiovascular disorders. In many of these diseases it is unclear how pathogenic changes begin or the cascade of events that ultimately cause disease. This is particularly true for atrial fibrillation (AF). The main risk factor for AF development is aging, and AF therapies are mostly focused on treatment rather than prevention. In large part this is due to our incomplete understanding of the factors that drive AF initiation and progression. Therefore, a more comprehensive understanding of the basic biology behind normal cardiac function and the molecular mechanisms behind pathological cascades will allow for the development of novel therapeutics that could prevent in addition to treat AF and other cardiovascular disorders.
We are particularly interested in a transmembrane heme containing signaling enzyme NADPH oxidase 5 (NOX5). NOX5 is activated by increases in intracellular calcium to produce a superoxide burst. While its physiological roles are still being uncovered, it’s been shown to be critical for monocyte differentiation into dendritic cells, differentiation and maturation of oligodendrocytes, sperm motility and viability, and vascular contraction. Additionally, it’s been implicated in cancers, diabetes, and cardiovascular disorders. Using a protocol to study heme insertion and subsequent activity of NOX5 we found that changes in intracellular heme levels, nitric oxide, and Hsp90 binding dynamically regulate NOX5 activity through controlling its heme saturation and oligomerization.
NOX5 in atrial fibrillation.
AF is characterized by rapid, irregular heartbeats and increases the risk for stroke, myocardial infarction, heart failure and death. Recently, NOX5 was identified as a regulator of vascular contraction, linking calcium and redox signaling, and a number of known NOX5 regulators including peptide hormones, kinases and intracellular Ca2+, have been shown to be dysregulated in AF. In preliminary studies we’ve found that NOX5 protein is found in tissue from the Left Atrial Appendage (LAA) from patients in sinus rhythm as well as patients in AF. We have also been able to study NOX5 activity in induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) and found that treatment with AngII and ET-1 increase superoxide production and that increased intracellular heme levels increases it further. Overall we are interested in understanding how NOX5 and changes in its expression and activity play a role in normal cardiomyocyte biology as well as the pathogenesis of atrial fibrillation. By coupling biochemistry, cell biology, and studies in patient derived cells and tissues, we can manipulate the system (pacing stress, changes in heme and NO exposure to iPSC-CMs) and study the downstream effects - alterations in NOX5 PTMs, protein partners, activity, expression, localization, and the global effects of these changes. Then using patient derived tissue we can test our hypotheses generated from the iPSC-CM system.
NOX5 protein partners
We have used two methods to probe the interactomes of a number of heme proteins including NOX5. The NOX5 protein partners pulled out of these MS screens show a strong enrichment for mitochondrial proteins, proteins associated with the actin cytoskeleton, RNA regulating proteins, and cellular chaperones. We are interested in understanding how these interactions contribute to physiological and pathological cellular responses not only to increase our basic biological understanding of the cell, but also to provide a path to the development of new therapeutics and biomarkers for disease diagnosis and treatment. We use a variety of techniques to study the role of these interactions in cells and how they drive cellular responses using model cell systems (HEK293 with and without NOX5 expression) and iPSC derived cardiomyocytes.
(Sweeny EA, Hunt AP, Batka AE, Schlanger S, Lehnert N, Stuehr DJ.) Free Radic Biol Med. 2021 08 20;172:252-263 PMID: 34139309 PMCID: PMC8355125 06/18/2021
(Sweeny EA, Schlanger S, Stuehr DJ.) Redox Biol. 2020 09;36:101656 PMID: 32738790 PMCID: PMC7394750 08/02/2020
(Dai Y, Sweeny EA, Schlanger S, Ghosh A, Stuehr DJ.) J Biol Chem. 2020 06 12;295(24):8145-8154 PMID: 32358060 PMCID: PMC7294094 05/03/2020
(Sweeny EA, Tariq A, Gurpinar E, Go MS, Sochor MA, Kan ZY, Mayne L, Englander SW, Shorter J.) J Biol Chem. 2020 02 07;295(6):1517-1538 PMID: 31882541 PMCID: PMC7008382 12/29/2019
(Tariq A, Lin J, Jackrel ME, Hesketh CD, Carman PJ, Mack KL, Weitzman R, Gambogi C, Hernandez Murillo OA, Sweeny EA, Gurpinar E, Yokom AL, Gates SN, Yee K, Sudesh S, Stillman J, Rizo AN, Southworth DR, Shorter J.) Cell Rep. 2019 08 20;28(8):2080-2095.e6 PMID: 31433984 PMCID: PMC6750954 08/23/2019
(Durie CL, Lin J, Scull NW, Mack KL, Jackrel ME, Sweeny EA, Castellano LM, Shorter J, Lucius AL.) Biophys J. 2019 05 21;116(10):1856-1872 PMID: 31027887 PMCID: PMC6531783 04/28/2019
(Sweeny EA, Singh AB, Chakravarti R, Martinez-Guzman O, Saini A, Haque MM, Garee G, Dans PD, Hannibal L, Reddi AR, Stuehr DJ.) J Biol Chem. 2018 09 14;293(37):14557-14568 PMID: 30012884 PMCID: PMC6139559 07/18/2018
(Guo L, Kim HJ, Wang H, Monaghan J, Freyermuth F, Sung JC, O'Donovan K, Fare CM, Diaz Z, Singh N, Zhang ZC, Coughlin M, Sweeny EA, DeSantis ME, Jackrel ME, Rodell CB, Burdick JA, King OD, Gitler AD, Lagier-Tourenne C, Pandey UB, Chook YM, Taylor JP, Shorter J.) Cell. 2018 04 19;173(3):677-692.e20 PMID: 29677512 PMCID: PMC5911940 04/21/2018
(Ghosh A, Garee G, Sweeny EA, Nakamura Y, Stuehr DJ.) Proc Natl Acad Sci U S A. 2018 02 06;115(6):E1117-E1126 PMID: 29358373 PMCID: PMC5819442 01/24/2018
(Gates SN, Yokom AL, Lin J, Jackrel ME, Rizo AN, Kendsersky NM, Buell CE, Sweeny EA, Mack KL, Chuang E, Torrente MP, Su M, Shorter J, Southworth DR.) Science. 2017 07 21;357(6348):273-279 PMID: 28619716 PMCID: PMC5770238 06/18/2017
(Burén S, Young EM, Sweeny EA, Lopez-Torrejón G, Veldhuizen M, Voigt CA, Rubio LM.) ACS Synth Biol. 2017 06 16;6(6):1043-1055 PMID: 28221768 PMCID: PMC5477005 02/22/2017
(Weaver CL, Duran EC, Mack KL, Lin J, Jackrel ME, Sweeny EA, Shorter J, Lucius AL.) Biochemistry. 2017 04 18;56(15):2071-2075 PMID: 28379007 PMCID: PMC5649362 04/06/2017