Maturational Breakdown of Mitochondria and Other Organelles in Reticulocytes

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the activesite volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "spacebased" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S-via 8S-toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9,11,14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5-and 15-lipoxygenases by sitedirected mutagenesis and appear to support the spacebased hypothesis of positional specificity. Lipoxygenases (LOXs)1 constitute a heterogeneous family of lipid-peroxidizing enzymes that catalyze the dioxygenation of free and/or esterified polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. In mammals LOXs are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-LOXs (1, 2). In contrast, plant physiologists prefer a linoleic acid-related enzyme nomenclature since arachidonic acid is only a minor fatty acid in plants. Mammalian 5-LOXs are key enzymes in the biosynthesis of leukotrienes, which are important mediators of inflammatory and anaphylactic disorders (3, 4). During the past 10 years, 5-LOX inhibitors and leukotriene receptor antagonists have been developed as anti-asthmatic drugs, and some of them are now available for prescription (5, 6). Mammalian 15-LOXs have been implicated in peroxisome proliferation activating receptor-␥-mediated cell signaling (7), in cell development and maturation (8, 9), as well as in the pathogenesis of atherosclerosis (10, 11). The intracellular activity of LOXs is regulated on pre-translational, translational, and posttranslational levels. Expression of the human 5-LOX is upregulated by transforming growth factor (12), and melatonin represses the 5-LOX pathway in B-lymphocytes (13). The interleukins-4 (14) and -13 (15) induce 15-LOX expression in monocyte/macrophages, and this regulatory process involves activation of the transcription factor STAT6 (16) as well as JAK2 and Tyk2 kinases (17). Transl...

Section: Lipoxygenases (Loxs)mentioning

confidence: 99%

Structural Basis for Lipoxygenase Specificity

Schwarz¹,

Walther²,

Anton³

et al. 2001

“…In mammals, LOXs are categorized with respect to their positional specificity of arachidonic acid oxygenation (2), but plant physiologists prefer a linoleic acid-related enzyme nomenclature (3). Mammalian LOXs are involved in the biosynthesis of inflammatory mediators, such as leukotrienes (4) and lipoxins (5), but have also been implicated in cell differentiation (6,7), carcinoma metastasis (8,9), and atherogenesis (10,11).…”

mentioning

confidence: 99%

Investigations into Calcium-dependent Membrane Association of 15-Lipoxygenase-1

Walther

Wiesner

Kühn

2004

Taken together these data suggest that 15-lipoxygenase-1 associates to biomembranes primarily via hydrophobic interactions between surface-exposed apolar amino acid side chains and membrane lipids. Calcium supports membrane binding probably by forming salt bridges between the negatively charged head groups of membrane phospholipids and acidic surface amino acids of the membrane contact plane and this interaction might contribute to overcome repulsive forces.

“…For example, 5-LOX 1 generates precursors for leukotrienes, products involved in inflammation and allergic responses (1). 12-Lipoxygenases, present in vascular endothelium, smooth muscle cells, platelets, and leukocytes (2, 3), contribute to vascular cell hypertrophy, proliferation, and hypertensive actions, while 15-LOX is involved in cell development and differentiation, particularly in reticulocytes where 15-LOX oxidation of mitochondrial phospholipids is a trigger for their degradation (2,4,5).A central pathogenic role for 15-LOX in atherosclerosis comes from multiple lines of evidence, in particular the colocalization of 15-LOX mRNA, enzymatic activity, and the relatively specific pattern of isomeric 15-LOX oxygenation products that have been detected in early human and rabbit lesions (6 -9). The accumulation and oxidation of low density lipoprotein (LDL) lipids by monocytes and the subsequent accumulation of oxidized lipids and foam cells in the vascular intima is a hallmark of early atherogenesis.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

15-Lipoxygenase Catalytically Consumes Nitric Oxide and Impairs Activation of Guanylate Cyclase

O'Donnell

Taylor²,

Parthasarathy

et al. 1999

Reactivation of E red , considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of ⅐ NO with 15-LOX inhibited ⅐ NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated ⅐ NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of ⅐ NO with lipoxygenase peroxyl radical intermediates will result in modulation of both ⅐ NO bioavailability and rates of production of lipid signaling mediators.Lipoxygenases are a family of ubiquitously expressed nonheme iron-containing enzymes that oxidize the unsaturated fatty acids arachidonate and linoleate to bioactive hydroperoxides and other metabolites (Scheme 1). For example, 5-LOX 1 generates precursors for leukotrienes, products involved in inflammation and allergic responses (1). 12-Lipoxygenases, present in vascular endothelium, smooth muscle cells, platelets, and leukocytes (2, 3), contribute to vascular cell hypertrophy, proliferation, and hypertensive actions, while 15-LOX is involved in cell development and differentiation, particularly in reticulocytes where 15-LOX oxidation of mitochondrial phospholipids is a trigger for their degradation (2,4,5).A central pathogenic role for 15-LOX in atherosclerosis comes from multiple lines of evidence, in particular the colocalization of 15-LOX mRNA, enzymatic activity, and the relatively specific pattern of isomeric 15-LOX oxygenation products that have been detected in early human and rabbit lesions (6 -9). The accumulation and oxidation of low density lipoprotein (LDL) lipids by monocytes and the subsequent accumulation of oxidized lipids and foam cells in the vascular intima is a hallmark of early atherogenesis. In vitro studies have shown