Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.

Spexin (SPX) is a neuropeptide identified recently by bioinformatic approach. At present not much is known about its biological actions, and comparative studies of SPX in nonmammalian species are still lacking. To examine the structure and function of SPX in fish model, SPX was cloned in goldfish and found to be highly comparable with its mammalian counterparts. As revealed by NMR spectroscopies, goldfish SPX is composed of an α-helix from Gln(5) to Gln(14) with a flexible NH2 terminus from Asn(1) to Pro(4), and its molecular surface is largely hydrophobic except for Lys(11) as the only charged residue in the helical region. In goldfish, SPX transcripts were found to be widely expressed in various tissues, and protein expression of SPX was also detected in the brain. In vivo feeding studies revealed that SPX mRNA levels in the telencephalon, optic tectum, and hypothalamus of goldfish brain could be elevated by food intake. However, brain injection of goldfish SPX inhibited both basal and NPY- or orexin-induced feeding behavior and food consumption. Similar treatment also reduced transcript expression of NPY, AgRP, and apelin, with concurrent rises in CCK, CART, POMC, MCH, and CRH mRNA levels in different brain areas examined. The differential effects of SPX treatment on NPY, CCK, and MCH transcript expression could also be noted in vitro in goldfish brain cell culture. Our studies for the first time unveil the solution structure of SPX and its novel function as a satiety factor through differential modulation of central orexigenic and anorexigenic signals.

Trapping of phenylacetaldehyde as a key mechanism responsible for naringenin's inhibitory activity in mutagenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine formation.

Chemical model reactions were carried out to investigate the mechanism of inhibition by a citrus flavonoid, naringenin, on the formation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine found in foods. GC-MS showed that naringenin dose dependently reduced the level of phenylacetaldehyde, a key intermediate on the pathway to the formation of PhIP. Subsequent LC-MS analyses of samples from a wide range of model systems consisting of PhIP precursors, including phenylalanine, glucose, and creatinine, suggested that naringenin scavenged phenylacetaldehyde via adduct formation. An isotope-labeling study showed that the postulated adducts contain fragment(s) of phenylalanine origin. Direct reaction employing phenylacetaldehyde and naringenin further confirmed the capability of naringenin to form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. Two of the adducts were subsequently isolated and purified. Their structure was elucidated by one- and two-dimensional NMR spectroscopy as 8- C-( E-phenylethenyl)naringenin (1) and 6- C-( E-phenylethenyl)naringenin (2), respectively, suggesting that C-6 and C-8 are two of the active sites of naringenin in adduct formation. These two adducts were also identified from thermally processed beef models, highlighting phenylacetaldehyde trapping as a key mechanism of naringenin to inhibit PhIP formation.

Domain organization of XAF1 and the identification and characterization of XIAP(RING) -binding domain of XAF1.

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1(NTD) ) followed by a middle domain (XAF1(MD) ), a 42-residue unstructured linker and a C-terminal domain (XAF1(CTD) ). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAP(RING) binding site (dissociation constant, K(d) , ∼18 µM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1(CTD) and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAP(RING) -binding domain of XAF1 (XAF1(RBD) ) in the present study. We have also mapped the interaction sites for XAF1(RBD) on XIAP(RING) by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1(RBD) /XIAP interaction is essential for the ubiquitination of GST-XAF1(RBD) fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1(RBD) was found to be substantially ubiquitinated by XIAP(RING) . Base on these observations, we speculate a possible role of XAF1(RBD) in targeting XAF1 for XIAP-mediated ubiquitination.