Original Contribution Subcellular localization of tyrosine-nitrated proteins is dictated by reactive oxygen species generating enzymes and by proximity to nitric oxide synthase Harry F.G. Heijnen a,b,c, * , Elly van Donselaar b,c , Jan W. Slot b,c , Diana M. Fries d,e , Beatrice Blachard-Fillion d , Roberto Hodara d , Richard Lightfoot d , Manuela Polydoro d , Dave Spielberg d , Leonor Thomson d,f , Elizabeth A. Regan g , James Crapo g , Harry Ischiropoulos d a Thrombosis and Hemostasis Laboratory, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands b Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands c Institute for Biomembranes, Utrecht, The Netherlands d Stokes Research Institute, Department of Biochemistry and Biophysics, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104, USA e Ministry of Health, Sao Paulo, SP, Brazil f Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay g National Jewish Medical and Research Center, Denver, CO 80206, USA Received 13 May 2005; revised 2 September 2005; accepted 2 September 2005 Available online 15 November 2005 Abstract Using high-resolution immuno-electron microscopy the steady-state subcellular distribution of tyrosine-nitrated proteins in different cells and tissues was evaluated. In quiescent eosinophils and neutrophils in the bone marrow intracellular nitrated proteins were mainly restricted to the peroxidase-containing secretory granules. The inducible nitric oxide synthase (iNOS) was expressed in the same granules. Proteins nitrated on tyrosine residues were also abundant in the cytosol of circulating erythrocytes. In the vasculature, nitrated proteins were mainly located in mitochondria and endoplasmic reticulum of the endothelial cells, fibroblasts, and smooth muscle cells. Endogenous nitrated proteins were also found in chondrocytes in cartilage, where it was typically associated with the cytoplasmic interface of the endoplasmic reticulum membrane. Nitrated proteins were also prominent in the peroxisomes of liver hepatocytes and of secretory cells in the lacrimal gland. Challenge of mouse dendritic cells with lipopolysaccharide induced iNOS protein expression in cytosol and peroxisomes and was associated with an increased 3- nitrotyrosine formation in cytosol, mitochondria, and peroxisomes. These data indicate that nitric oxide-dependent protein tyrosine nitration is a physiologically relevant process localized within specific subcellular compartments in close proximity to iNOS and to enzymes capable of peroxidative chemistry and reactive oxygen species production. D 2005 Elsevier Inc. All rights reserved. Keywords: Immuno-electron microscopy; Nitric oxide; Nitrotyrosine; Superoxide; Peroxidases; Free radicals The formation of nitric oxide (NO) in biological systems has been intimately linked with the discovery of a number of NO- dependent posttranslational protein modifications that could regulate protein function and/or be employed as transducers of nitric oxide signaling [1,2]. One of the nitric oxide-mediated protein modifications is the nitration of one of the two equivalent carbons (carbon 3) in the aromatic ring of tyrosine residues. This results in the formation of an unusual amino acid, 3-nitrotyrosine. Because the formation of 3-nitrotyrosine requires the formation of higher oxides of nitric oxide, protein nitration has been mostly associated with pathological condi- tions of oxidative stress [3–5]. Indeed, immunohistochemical and analytical methodologies have been employed to detect and quantify nitrated proteins in several major human diseases [3,4]. The advent of proteomics has dramatically and signif- icantly expanded these data by identifying specific proteins in the context of disease conditions that are modified by nitration 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.006 * Corresponding author. Thrombosis and Hemostasis Laboratory, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Nether- lands. Fax: +31 30 2541797. E-mail address: h.f.g.heijnen@azu.nl (H.F.G. Heijnen). Free Radical Biology & Medicine 40 (2006) 1903 – 1913 www.elsevier.com/locate/freeradbiomed