Identification of oxidative modifications of hemopexin and their predicted physiological relevance.

Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis, and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-hemopexin is vulnerable to inactivation by reactive nitrogen (RNS) and oxygen species (ROS) that covalently modify amino acids. The resultant nitration of amino acids is considered a specific effect reflecting biological events. Using LC-MS, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of human hemopexin, which was similarly nitrated in rabbit and rat hemopexins. Immunoblotting and selective reaction monitoring were used to quantify tyrosine nitration of in vivo samples and when hemopexin was incubated in vitro with nitrating nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by hemopexin declined as tyrosine nitration proceeded in vitro Three nitrated tyrosines reside in the heme-binding site of hemopexin, and we found that one, Tyr-199, interacts directly with the heme ring D propionate. Investigating the oxidative modifications of amino acids after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro, we identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of hemopexin. Importantly, three of the four modified tyrosines, some of which have more than one modification, cluster in the heme-binding site, supporting a hierarchy of vulnerable amino acids. We propose that during inflammation, apo-hemopexin is nitrated and oxidated in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing hemopexin's protective extracellular antioxidant function.

A One Health overview, facilitating advances in comparative medicine and translational research.

TABLE OF CONTENTS: A1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17 Polycystic kidney disease: cellular mechanisms to emerging therapiesJames P. CalvetA18 The domestic cat as a large animal model for polycystic kidney diseaseLeslie A. Lyons, Barbara GandolfiA19 The support of basic and clinical research by the Polycystic Kidney Disease FoundationDavid A. BaronA20 Using naturally occurring large animal models of human disease to enable clinical translation: treatment of arthritis using autologous stromal vascular fraction in dogsMark L. WeissA21 Regulatory requirements regarding clinical use of human cells, tissues, and tissue-based productsDebra A. WebsterA22 Regenerative medicine approaches to Type 1 diabetes treatmentFrancis N. KaranuA23 The zoobiquity of canine diabetes mellitus, man's best friend is a friend indeed-islet transplantationEdward J. RobbA24 One Medicine: a development model for cellular therapy of diabetesRobert J. Harman.

Ceestatin, a novel small molecule inhibitor of hepatitis C virus replication, inhibits 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

BACKGROUND: Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library. METHODS: To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatin's anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. CONCLUSIONS: Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.