Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Abstract

Hepatocytes are capable of repeated inducible NO synthase (iNOS) expression, which occurs under inflammatory and stress conditions. This iNOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of iNOS in these cells, we characterized the iNOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of iNOS in cytokine-stimulated hepatocytes and that the iNOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released iNOS to the soluble fraction. iNOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted iNOS exhibits a lower specific activity than iNOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric iNOS without a greater relative distribution of iNOS to the peroxisomal pool. Thus, iNOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric iNOS. iNOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme. subcellular localization ͉ inflammatory and stress conditions ͉ hepatocytes ͉ peroxisomes N itric oxide (NO) has critical signaling functions within cells but can also lead to cell dysfunction or toxicity. One mechanism for both regulating and localizing NO production is through distribution of NO synthase (NOS) to structures within the cells. For example, the association of endothelial NOS (eNOS) with caveolin localizes eNOS within structures in the plasma membrane known as caveolae and is a mechanism for regulating eNOS activity (1). The physical interaction of neuronal NOS (nNOS) with other proteins containing PDZ domains localizes this isoform to synaptic junctions in brain and motor endplates in skeletal muscle (2). Comparatively little is known about the posttranslational modifications that regulate inducible NOS (iNOS) distribution and function. In murine macrophages, iNOS has been shown to be both phosphorylated and ubiquitinated (3) and also associates with Rac1 and Rac2 as a mechanism to increase enzyme activity (4). We have previously shown that iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro (5) and in vivo (5, 6); however, the structure and function of this cellular pool of iNOS is unknown.Proteins that localize to peroxisomes function in hydrogen peroxide breakdown, lipid metabolism, and amine synthesis (7). Peroxisomes are single-membrane organelles of 0.2-1.0 m in size that do not require protein unfolding or disruption of multimeric complexes before protein import (7). These organelles are particularly abundant in hepatocytes, constituting Ϸ1% of tota...

Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants

“…Several NADP-dehydrogenases can produce NADPH (Corpas et al, 1998, while the presence of calmodulin in plant peroxisomes has been also demonstrated (Yang and Poovaiah, 2002) being both essential elements for this activity. These results could be important, particularly given that inducible NOS has also been reported in peroxisomes of rat hepatocytes (Stolz et al, 2002;Loughran et al, 2005).…”

Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

“…Furthermore, even if a protein's sequence is not 100% identical to the canonical PTS sites, other enzymes have been described that target to the peroxisome with similar, but nonconventional PTS sequences; these include acetoacetyl-CoA thiolase, 39 alanine:glyoxylate aminotransferase, 40 isopentenyl diphosphate dimethylallyl diphosphate isomerase, 41 and iNOS. 27,42 In fact, very large protein oligomers lacking PTS motifs have been shown to piggy-back onto other conventional peroxisomal proteins and gain entry into the peroxisomal matrix in their native three-dimensional configuration. 43 Specifically, the primarily hydrophobic ␤-domain of vHL contains two critical hydrophilic residues (His-115 and Ser-111), which must hydrogen bond with either hydroxyproline or H 2 O molecules.…”

“…In the case of iNOS, a nonconventional peroxisomal enzyme in hepatocytes, the fraction of iNOS sequestered in peroxisomes is an inactive monomer, perhaps protecting the cell from incompetent enzyme. 42 On the other hand, phytanoyl Co-A hydroxylase is a classic PTS2-containing enzyme that leads to Refsum's disease if defective. 56 Not only is phytanoyl Co-A hydroxylase active in peroxisomes, it is actually a member of the same O 2 -, Fe 2ϩ -, and 2-oxogluterate-dependent oxygenase family as the HIF hydroxylases; therefore, within the peroxisomes there may exist a medium containing the co-factors necessary for PHD and FIH-1 activity.…”

NF‐κB in liver diseases: a target for drug therapy

“…Chaisson et al (2002) have shown that TNF-α activates NF-κB in many cells after intraperitoneal injection. Inducible nitric oxide synthase (iNOS) transcription is also induced by TNF-α and IL-6 through NF-κB activity (Shin et al, 2003); this occurs mainly in hepatocytes just after partial hepatectomy and before cell proliferation (Loughran et al, 2005). The most important growth factor involved in hepatocyte proliferation, HGF, signals through its receptor c-Met, a transmembrane tyrosine kinase protein product of the proto-oncogene with the same name (Huh et al, 2004).…”