Abstract-Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, L-arginine. However, the high K m of arginase (Ͼ1 mmol/L) compared with NOS (2 to 20 mol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for L-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its K m value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its K m decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for L-arginine and contribute to endothelial dysfunction in the aging cardiovascular system. (Circ Res. 2007;101:692-702.)Key Words: arginase Ⅲ NO synthase Ⅲ S-nitrosylation Ⅲ aging A ging is accompanied by impaired endothelial function caused by reduced NO bioavailability. Arginase, the final enzyme of the urea cycle, uses L-arginine as a substrate 1-4 and reciprocally regulates NO synthase (NOS) by substrate depletion. [5][6][7][8] Increased arginase activity, therefore, leads to diminished production of NO. 6,[9][10][11][12] In vascular tissue, this further affects 2 important functions of NO: (1) cGMPdependent signaling and (2) modulation of protein function through S-nitrosylation. Several studies have demonstrated reciprocal regulation of arginase and NOS, where inhibition of arginase leads to increased NO activity. 7,8,13,14 Conversely, upregulation of arginase functionally inhibits NOS activity and contributes to the pathophysiology of several disease processes, 8,[15][16][17][18] including age-related vascular dysfunction. 5,6 The high K m value of human arginase for L-arginine (Ͼ1 mmol/L 19 ) is significantly higher than that of NOS (2 to 20 mol/L 20 ) and suggests that there should not be a direct competition for L-arginine. However, the role of arginase in regulating NOS activity through substrate depletion is now well detailed, as discussed above.Given the interaction between NOS and arginase signaling, we hypothesized that S-nitrosylation of arginase1 might be an important posttranslational modification mechanism that regulates its activity. The protein sequence for human arginase1 contains 3 cysteines, C45, C168, and C303, with C303 being very close...

Materials and Methods Cell Transfections and ImmunoblottingCysteines 65

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-inducd apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interferencemediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.

Objective-Intercellular adhesion molecule-1 (ICAM-1) is upregulated rapidly on endothelial cells during ischemiareperfusion (I-R) and mediates tissue leukocyte accumulation. The ICAM-1 proximal promoter contains a signal transducer and activator of transcription (Stat) binding motif (gamma-interferon activation site [GAS] sequence), which flanks a specificity protein 1 (Sp1) binding site. We examined the roles of Stat and Sp1 in the regulation of ICAM-1 after myocardial I-R. Methods and Results-Open-chest anesthetized rats underwent coronary artery occlusion for 35 minutes and reperfusion for 0 to 240 minutes. Stat became activated within 15 minutes after reperfusion, primarily in vascular endothelial cells; the activated Stat protein was identified as Stat3 (␣-isoform). After phosphorylation on serine 727 (p-S727), Stat3␣ was found in association with the transcriptional regulator Sp1, and the complex bound to an ICAM-1-GAS probe. ICAM-1 expression increased after I-R and lagged shortly behind Stat3␣ activation. In cultured human umbilical vein endothelial (HUVE) cells, activation of Stat3␣ after hypoxia-reoxygenation (H-R) was dependent on the small GTPase Rac1. Transfection of a dominant-negative Stat3 (Y705F) adenovirus or a GAS decoy oligonucleotide reduced ICAM-1 mRNA expression after H-R. Using a reporter gene transfected into HUVE cells, mutation of the GAS element in the ICAM-1 promoter resulted in reduced transcriptional activity after H-R. Sp1 coimmunoprecipitated with p-S727 Stat3 during H-R, and Sp1 or Stat3␣ interfering RNA markedly reduced ICAM-1 mRNA expression. Key Words: adhesion molecule Ⅲ signal transduction Ⅲ ischemia-reperfusion Ⅲ myocardium Ⅲ endothelial cell I ntercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein that is highly expressed in vascular endothelial cells, promotes leukocyte activity in a variety of inflammatory reactions, and plays an important role in mediating neutrophil adherence and tissue injury during reperfusion after ischemia. 1,2 The ICAM-1 proximal promoter contains a signal transducer and activator of transcription 1 (Stat1)/Stat3 binding motif (GAS sequence or palindromic interferon response element), which flanks a specificity protein 1 (Sp1) binding site and a promoter cis-acting sequence (TATAA) box. Reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ) and superoxide, and proinflammatory cytokines such as interleukin-6 (IL-6) and interferon-␥ (IFN-␥), are known to induce transcriptional complexes that bind to the ICAM-1 GAS sequence. [3][4][5] Mutation or deletion of this element decreases ICAM-1 promoter activity. 4,5 In the past 10 years, Stat proteins have been studied intensively as cellular transcriptional regulators. Various Stat proteins, including Stat1 and Stat3, have been reported to be activated in ischemia-reperfusion (I-R). 6,7 However, the role of Stats in I-R is unclear because Stats appear to mediate a broad range of seemingly conflicting processes, including apoptosis, survival pathways, proliferation, angiog...

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Syntrophins are a family of cytoplasmic membrane-associated adaptor proteins, characterized by the presence of a unique domain organization comprised of a C-terminal syntrophin unique (SU) domain and an N-terminal pleckstrin homology (PH) domain that is split by insertion of a PDZ domain. Syntrophins have been recognized as an important component of many signaling events, and they seem to function more like the cell's own personal 'Santa Claus' that serves to 'gift' various signaling complexes with precise proteins that they 'wish for', and at the same time care enough for the spatial, temporal control of these signaling events, maintaining overall smooth functioning and general happiness of the cell. Syntrophins not only associate various ion channels and signaling proteins to the dystrophin-associated protein complex (DAPC), via a direct interaction with dystrophin protein but also serve as a link between the extracellular matrix and the intracellular downstream targets and cell cytoskeleton by interacting with F-actin. They play an important role in regulating the postsynaptic signal transduction, sarcolemmal localization of nNOS, EphA4 signaling at the neuromuscular junction, and G-protein mediated signaling. In our previous work, we reported a differential expression pattern of alpha-1-syntrophin (SNTA1) protein in esophageal and breast carcinomas. Implicated in several other pathologies, like cardiac dys-functioning, muscular dystrophies, diabetes, etc., these proteins provide a lot of scope for further studies. The present review focuses on the role of syntrophins in membrane targeting and regulation of cellular proteins, while highlighting their relevance in possible development and/or progression of pathologies including cancer which we have recently demonstrated.

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