Activation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (

The phosphatidylinositol 3-kinase/Akt pathway plays a critical role in oncogenesis, and dysregulation of this pathway through loss of PTEN suppression is a particularly common phenomenon in aggressive prostate cancers. The mammalian target of rapamycin (mTOR) is a downstream signaling kinase in this pathway, exerting prosurvival influence on cells through the activation of factors involved in protein synthesis.

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl-N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in Vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in Vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in ViVo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J 3 -isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.Eicosapentaenoic acid (EPA, C20:5n-3) 5 and docosahexaenoic acid (DHA, C22:6n-3) are two major components of fish oil. Epidemiological studies and randomized controlled trials have demonstrated that n-3 fatty acid supplementation can reduce sudden cardiac death, nonfatal stroke, and decrease progression of arteriosclerosis (1-3). Dietary supplementation with fish oil also shows promise for suppressing progression of neurodegenerative diseases (4), neuropsychiatric disorders (5), human immunodeficiency virus (6), and diseases of the retina (7). Thus, identifying the molecular mechanism(s) responsible for n-3 fatty acid mitigation of these diverse diseases represents an important and intriguing question.Oxidative stress is a common factor in the etiology of the diseases impacted by DHA and EPA (1, 8 -17). These n-3 fatty acids are very susceptible to free radical oxidation, exceeding that of arachidonic acid (AA) (18 -20). Yet, recent studies have shown that EPA and DHA supplementation reduced urinary F 2 -isoprotane levels, a marker for oxidative stress, as well as enhanced cellular antioxidant defense systems (21-24). Although a reduction of F 2 -isoprostane levels can be attributed, in part, to a decrease in membrane AA content (20,25), the relationship between EPA/ DHA oxidation versus EPA/DHA-mediated induction of antioxidant responses is not well understood.Non-enzymatic free-radical peroxidation of AA results in the formation of multiple stereoisomers of prostaglandin H 2 -like bicyclic endoperoxides that can undergo thiol-mediated reduction to form prostaglandin-like compounds termed F 2 -isoprostanes (F 2 -IsoPs) or rearrangement and reduction to form E 2 -IsoPs, D 2 -IsoPs, and isothromboxanes. E 2 /D 2 -IsoPs can then dehydrate to highly reactive A 2 /J 2 -isoprostanes containing cyclopentenone rings. These compounds readily adduct thiol groups on proteins and have been shown to induce Nrf2/ ARE-directed gene expression (26).NF-E2-related factor 2, Nrf2, is a master transcription factor ...

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC 50 , K D (isotherm), K D (dissociation rate), and K D ( 1 ⁄2 EC 50 ), respectively, were the following: sildenafil (3.7 Ϯ 1.4, 4.8 Ϯ 0.80, 3.7 Ϯ 0.29, and 11.7 Ϯ 0.70 nM), tadalafil (1.8 Ϯ 0.40, 2.4 Ϯ 0.60, 1.9 Ϯ 0.37, and 2.7 Ϯ 0.25 nM); and vardenafil (0.091 Ϯ 0.031, 0.38 Ϯ 0.07, 0.27 Ϯ 0.01, and 0.42 Ϯ 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil Ͼ Ͼ tadalafil Ͼ sildenafil. Binding of each 3 H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds.