XIAP Is a copper binding protein deregulated in Wilson's disease and other copper toxicosis disorders

X-linked inhibitor of apoptosis (XIAP), known primarily for its caspase inhibitory properties, has recently been shown to interact with and regulate the levels of COMMD1, a protein associated with a form of canine copper toxicosis. Here, we describe a role for XIAP in copper metabolism. We find that XIAP levels are greatly reduced by intracellular copper accumulation in Wilson's disease and other copper toxicosis disorders and in cells cultured under high copper conditions. Elevated copper levels result in a profound, reversible conformational change in XIAP due to the direct binding of copper to XIAP, which accelerates its degradation and significantly decreases its ability to inhibit caspase-3. This results in a lowering of the apoptotic threshold, sensitizing the cell to apoptosis. These data provide an unsuspected link between copper homeostasis and the regulation of cell death through XIAP and may contribute to the pathophysiology of copper toxicosis disorders.     

Oxygenation of omega-3 fatty acids by human cytochrome P450 4F3B: effect on 20-hydroxyeicosatetraenoic acid production

Cytochrome P450 (CYP) omega-oxidases convert arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), a lipid mediator that modulates vascular tone. We observed that a microsomal preparation containing recombinant human CYP4F3B, which converts AA to 20-HETE, converted eicosapentaenoic acid (EPA) to 20-OH-EPA. Likewise, docosahexaenoic acid (DHA) was converted to 22-OH-DHA, indicating that human CYP4F3B also can oxidize 22-carbon omega-3 fatty acids. Consistent with these findings, addition of 0.5-5 microM EPA, DHA or omega-3 docosapentaenoic acid (DPA) to incubations containing 0.5 microM [3H]AA inhibited [3H]20-HETE production by 15-65%. [3H]20-OH-EPA was rapidly taken up by COS-7 cells, and almost all of the incorporated radioactivity remained as unmodified 20-OH-EPA. The 20-OH-EPA stimulated luciferase activity in COS-7 cells that express peroxisome proliferator-activated receptor alpha, indicating that this EPA metabolite may function as a lipid mediator. These findings suggest that some functional effects of omega-3 fatty acid supplementation may be due to inhibition of 20-HETE formation or the conversion of EPA to the corresponding omega-oxidized product.     

Imidazolylpyrimidine based CXCR2 chemokine receptor antagonists

An imidazolylpyrimidine was identified in a CXCR2 chemokine receptor antagonist screen and was optimized for potency, in vitro metabolic stability, and oral bioavailability. It was found that subtle structural modification within the series affected the oral bioavailability. Potent and orally available CXCR2 antagonists are herein reported.     

Hetaryl imidazoles: a novel dual inhibitors of VEGF receptors I and II

A novel potent derivatives of hetaryl imidazoles were described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Several compounds display VEGFR-2 inhibitory activity reaching IC(50)<100 nM in both enzymatic and cellular assays. The compounds also inhibit the related tyrosine kinase, VEGFR-1. By controlling the substitution pattern on the 5-carboxamido functionality, both dual and specific VEGFR-2 thiazoles were identified.     

TWEAK is a novel arthritogenic mediator

TNF-like weak inducer of apoptosis (TWEAK) is a TNF family member with pleiotropic effects on a variety of cell types, one of which is the induction of proinflammatory cytokines by synovial fibroblasts derived from rheumatoid arthritis (RA) patients. In this study, we report that the serum TWEAK level was dramatically elevated during mouse collagen-induced arthritis (CIA) and blocking TWEAK by a neutralizing mAb significantly reduced the clinical severity of CIA. Histological analyses also revealed that TWEAK inhibition diminished joint inflammation, synovial angiogenesis, as well as cartilage and bone erosion. Anti-TWEAK treatment proved efficacious when administered just before the disease onset but not during the priming phase of CIA. Consistent with this, TWEAK inhibition did not affect either cellular or humoral responses to collagen. In contrast, TWEAK inhibition significantly reduced serum levels of a panel of arthritogenic mediators, including chemokines such as MIP-1beta (CCL-4), lymphotactin (XCL-1), IFN-gamma-inducible protein 10 (IP-10) (CXCL-10), MCP-1 (CCL-2), and RANTES (CCL-5), as well as the matrix metalloprotease-9. Exploring the possible role of the TWEAK/Fn14 pathway in human RA pathogenesis, we showed that TWEAK can target human primary chondrocytes and osteoblast-like cells, in addition to synovial fibroblasts. We further demonstrated that TWEAK induced the production of matrix metalloproteases in human chondrocytes and potently inhibited chondrogenesis and osteogenesis using in vitro models. These results provide evidence for a novel cytokine pathway that contributes to joint tissue inflammation, angiogenesis, and damage, as well as may inhibit endogenous repair, suggesting that TWEAK may be a new therapeutic target for human RA.     

Phytosterol biosynthesis pathway in Mortierella alpina

The Zygomycetes fungus Mortierella alpina was cultured to growth arrest to assess the phytosterol biosynthesis pathway in a less-advanced fungus. The mycelium was found to produce 13 sterols, but no ergosterol. The sterol fractions were purified to homogeneity by HPLC and their identifies determined by a combination of GC-MS and 1H NMR spectroscopy. The principal sterol of the mycelium was cholesta-5, 24-dienol (desmosterol) (83%), with lesser amounts of 24beta-methyl-cholesta-5,25(27)-dienol (codisterol) (2%), 24-methyldesmosterol (6%), 24(28)-methylene cholesterol (3%) and lanosterol (3%) and several other minor compounds (3%). The total sterol accounted for approximately 0.07% of the mycelial dry wt. Mycelium fed methionine-methyl-2H3 for 6 days, generated 3 2H-24-methyl(ene) sterols, [C28-2H2]24(28)-methylenecholesterol, [C28-2H3]24-methylcholesta-5,24-dienol and [C28-2H3]24beta-methyl-cholesta-5,25(27)-dienol. The formation of the 24-methyl sterols seems to be catalyzed by the direct methylation of a common Delta24-acceptor sterol thereby bypassing the intermediacy of an isomerization step for rearrangement of the Delta24(28)-bond to Delta25(25)-position as operates in Ascomycetes fungi and all plants.     

The E3 Ubiquitin Ligase cIAP1 Binds and Ubiquitinates Caspase-3 and -7 via Unique Mechanisms at Distinct Steps in Their Processing

Inhibitor of apoptosis (IAP) proteins are widely expressed throughout nature and suppress cell death under a variety of circumstances. X-linked IAP, the prototypical IAP in mammals, inhibits apoptosis largely through direct inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once thought to also inhibit caspases, but more recent studies have suggested otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the proteolytic activities of effector caspases on fluorogenic or endogenous substrates. However, cIAP1 does bind to caspase-3 and -7 and does so, remarkably, at distinct steps prior to or following the removal of their prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif, AKPD, on the N terminus of the large subunit of fully mature caspase-7, whereas cIAP1 bound to partially processed caspase-3 in a manner that required its prodomain and cleavage between its large and small subunits but did not involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier protein/ubiquitin-conjugating enzymes), and in the case of caspase-3, differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric caspase-3 (bearing the AKPD motif) were degraded in vivo in a proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least in part, by facilitating the ubiquitination and turnover of active effector caspases in cells.Apoptosis is a programmed form of cell death that is generally executed through the activation of caspases,² cysteine proteases that exhibit an almost absolute preference for cleavage after aspartate residues. Caspases are synthesized as single-chain zymogens, containing a prodomain, as well as large and small subunits that include residues required for substrate recognition and cleavage (1). During death receptor or mitochondria-dependent apoptosis, the long prodomain-containing initiator caspase-8/10 and -9 are recruited via their adapter proteins, Fas-associated death domain and apoptotic protease-activating factor-1 (Apaf-1), to multimeric caspase-activating complexes known as the death-inducing signaling complex and the apoptosome, respectively (1, 2). In the latter case, mitochondrial outer membrane permeabilization (MOMP) is required to mediate the release of cytochrome c from the intermembrane space into the cytosol, where it stimulates dATP/ATP-dependent oligomerization of Apaf-1 into the apoptosome (2). Once recruited, all initiator caspases are concentrated within their respective complexes and are thought to be activated as a result of dimerization, with concomitant autocatalytic cleavage of the activation loops that separate their large and small subunits (1). However, unlike caspase-8 and -10, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity, so that in addition to promoting dimerization, the apoptosome may also induce conformational changes in caspase-9 that are necessary for its activation (3–6).In contrast to initiator caspases, effector caspases, such as caspase-3 and -7, contain short prodomains and exist normally as latent dimers, wherein their activation loops sterically hinder substrate access and hold the substrate binding pocket in an inactive conformation (1). Effector caspases are directly activated by caspase-8, -9, and -10, and following cleavage of caspase-3 between its large and small subunits, the two-chain p20/p12 form becomes a catalytically active heterotetramer and undergoes subsequent autocatalytic processing between its prodomain and large subunits to generate the fully mature p17/p12 form of the enzyme (7). Similarly, procaspase-7 is also activated following cleavage of its activation loop to generate its two-chain p22/p12 form; however, it remains unclear whether removal of its prodomain in cells (to generate its p19/p12 form) is accomplished primarily via autocatalysis, active caspase-3, or perhaps by serine proteases at a non-aspartate residue (8, 9). Caspase-3 and -7 exhibit significant sequence and structural homology, differing primarily in their short prodomains. Despite this fact, caspase-3 processes a wider array of protein substrates during apoptosis and is largely responsible for dismantling the cell (10). Thus, interesting questions remain regarding the physiological roles of caspase-7, whether caspase-7 activity is differentially regulated compared with caspase-3, and what structural features determine (and in some cases limit) its substrate specificity.Given the devastating consequences of unfettered caspase activation, cells have evolved mechanisms to regulate caspase activity. For example, IAPs, originally identified in baculoviruses, possess one or more baculovirus IAP repeat (BIR) domains, and at least one of the eight family members, XIAP, selectively inhibits the activities of caspase-9, -3, and -7 (1, 11). Mechanistically, the BIR3 domain in XIAP binds to an exposed IBM on the N terminus of the small subunit of processed caspase-9, situated directly above the active site, and limits the access of substrates (12, 13). By contrast, the linker region (located between the BIR1 and BIR2 domains in XIAP) lies across the active sites of caspase-3 and -7 and binds in a reverse orientation to substrates, thereby preventing cleavage of the linker while simultaneously preventing the access of substrates (14, 15). The BIR2 domain then stabilizes the linker-caspase-3 (and linker-caspase-7) interactions further by binding to an exposed IBM on the N terminus of the small subunit in the adjacent caspase dimer (14, 16). Importantly, IAP antagonists, such as Smac/DIABLO and Omi/HtrA2, are normally sequestered to the intermembrane space of mitochondria and are released (along with cytochrome c) into the cytoplasm during apoptosis. As IAP antagonists also possess IBMs, they bind to BIR domains and prevent or relieve the inhibition of caspases by IAPs (1).Previously, two additional IAP family members, cIAP1 and cIAP2, were also thought to inhibit caspases, but more recent studies suggest that these IAPs bind but do not inhibit caspases (17–19). Nevertheless, various studies have shown that cIAPs can protect cells from apoptosis, are overexpressed or mutated in some cancers, and can promote tumorigenesis (20–25), raising questions as to how these IAPs inhibit cell death or whether they have additional functions (26). XIAP, cIAP1, and cIAP2 possess C-terminal RING zinc finger domains with E3 ubiquitin (Ub) ligase activities capable of catalyzing the ubiquitination and subsequent proteasomal degradation of cellular targets, including themselves (27, 28). Moreover, cIAPs have been shown to ubiquitinate several factors, including TNF receptor-associated factor 2, the serine/threonine kinase NIK, receptor-interacting protein 1, and the IAP antagonist Smac (29–34). However, although there is some evidence to support a direct role for ubiquitination in the regulation of effector caspases by XIAP (35, 36), the role of cIAPs in this process remains unclear, particularly in vivo. We demonstrate herein that cIAP1 binds to caspase-3 and -7 at unique steps in their processing, prior to or following the removal of their prodomains, respectively. Moreover, rather than directly inhibiting these effector caspases, cIAP1 ubiquitinates them and targets them for proteasome-dependent degradation, thereby suppressing apoptosis.