Human arginase I is a potential target for therapeutic intervention in diseases linked to compromised L-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH, K d ؍ 5 nM) and S-(2-boronoethyl)-L-cysteine (BEC, Kd ؍ 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme-inhibitor complexes at 1.29-and 1.94-Å resolution determined from crystals twinned by hemihedry. The ultrahighresolution structure of the human arginase I-ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate-manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor ␣-amino and ␣-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state for L-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group of L-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.boronic acid ͉ metalloenzyme ͉ protein crystallography A rginase is a trimeric binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea (1-3). Two isozymes have been identified in mammals: arginase I catalyzes the final cytosolic step of the urea cycle in liver, and arginase II is a mitochondrial enzyme that functions in Larginine homeostasis in nonhepatic tissues. Notably, arginase I is also expressed in certain nonhepatic tissues where it, too, can function in L-arginine homeostasis. For example, arginase I may regulate substrate L-arginine bioavailability to NO synthase in the immune response. Macrophage arginase I and NO synthase are reciprocally regulated at the level of transcription: NO synthase is induced by T-helper type 1 (TH1) cytokines, and arginase I is induced by T-helper type 2 (TH2) cytokines (4-7). As a modulator of NO-dependent macrophage cytotoxicity, arginase I is implicated in the regulation of macrophage activity in wound healing (8) and the suppression of the tumoricidal activity of macrophages (9) and T cells (10). Notably, arginase I is very highly up-regulated in the murine spinal cord during experimental autoimmune encephalomyelitis, an animal model for human multiple sclerosis (11), and it is up-regulated in the inflammatory regions of the asthmatic lung (12)(13)(14).Arginase I in the immune response is also implicated in cancer biology: arginase I is significantly up-regulated and promotes tumor cell growth in breast cancer (15, 16) and colorectal cancer (17). Rodriguez et a...
Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/ hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38c MAP kinase (SAPK3/p38c) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38c-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.
Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERa)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kB, were both inhibited by RES. The patterns for NFkB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kB and calpain protease activity. Therefore, Bcl-2 and NF-kB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells. ' 2005 Wiley-Liss, Inc.Key words: resveratrol; caspase; Bcl-2; NF-kB; apoptosis Among natural compounds with beneficial effects on human health, RES (3,4 0 ,5-trihydroxystilbene) has attracted considerable interest. This molecule, present at relevant concentrations in red wine, 1 has been associated with a lower incidence of cardiovascular disease. Different studies have also suggested a beneficial effect of RES in cancer since it inhibits proliferation and promotes death in tumor cell lines of different origins, 2-4 and, in vivo, suppresses the formation of skin 5 and mammary gland 6 tumors in rodent models of carcinogenesis.We, as well as other laboratories, have found that the ability of RES to inhibit cell viability and proliferation in the human breast cancer cell lines MCF-7 and MDA-MB-231 was unrelated to ERa status. 7,8 However, apoptotic cell death was present only in ERapositive MCF-7 and involved cell-specific regulation of the G 1 /S and G 2 /M transitions of the cell cycle. 2,8 RES properties are related to ERa since this compound has estrogenic or antiestrogenic activities depending on its concentration and the phenotype of the target cell. 9,10 ERa, in addition to its nuclear role as a transcription factor, is involved in regulating the PI3K pathway, which controls cell growth, proliferation and apoptosis. [11][12][13] In MCF-7 cells, RES induced a biphasic pattern of PI3K activity that increased at low concentrations and decreased at high concentrations. Activation of downstream PI3K effectors PKB/AKT and GSK-3 closely followed the pattern of PI3K activity. 14 The ...
SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.
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