Distribution and expression of GnRH 1, kiss receptor 2, and estradiol α and ß receptors in the anterior brain of females of Chirostoma humboldtianum.

Reproduction in vertebrates is a complex process regulated by many hormones, and by paracrine factors and their receptors. This study aimed to examine the expression of pjGonadotropin-releasing hormone (GnRH 1), the kisspeptin receptor 2 (kissr2), and estradiol receptors α and ß (ER α and ER ß) during different stages of the sexual cycle and their distribution within the anterior brain of females of Chirostoma humboldtianum. Among these molecules, the kissr2 showed the maximal variation in expression, while GnRH 1 showed minimal variation of expression, and ERß and ERα had intermediate variation of expression. The distribution of these molecules in the anterior brain was consistent with their levels of expression; kissr2 was widely distributed throughout the telencephalon and diencephalon, while ER and GnRH 1 showed more restricted distributions. No coexpression of kissr2 and ER in GnRH 1ergic neurons, suggesting that regulation of this GnRH variant is indirectly mediated by kisspeptin and estradiol.

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding.

The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial-mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transport-deficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc.

Proteomic studies on protein modification by cyclopentenone prostaglandins: expanding our view on electrophile actions.

Cyclopentenone prostaglandins (cyPG) are lipid mediators that participate in the mechanisms regulating inflammation and tumorigenesis. cyPG are electrophilic compounds that act mainly through the covalent modification of cellular proteins. The stability of many cyPG-protein adducts makes them suitable for proteomic analysis. Indeed, methodological advances in recent years have allowed identifying many cyPG targets, including components of pro-inflammatory transcription factors, cytoskeletal proteins, signaling kinases and proteins involved in redox control. Insight into the diversity of cyPG targets is providing a better understanding of their mechanism of action, uncovering novel links between resolution of inflammation, proliferation and redox regulation. Moreover, identification of the target residues has unveiled the selectivity of protein modification by these electrophiles, providing valuable information for potential pharmacological applications. Among the challenges ahead, the detection of proteins modified by endogenous cyPG and the quantitative aspects of the modification require further efforts. Importantly, only a few years after the appearance of the first proteomic studies, research on cyPG targets is yielding new paradigms for redox and electrophilic signaling.

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification.

The cyclopentenone prostaglandin (cyPG) PGA(1) displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA(1) derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA(1) (PGA(1)-biotinamide, PGA(1)-B), to establish its validity to identify cyPG-protein interactions of potential therapeutic interest. PGA(1) and PGA(1)-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA(1)-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA(1)-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA(1)-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA(1) that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.

Selective binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid to peroxisome proliferator-activated receptor gamma allows ligand identification and characterization.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARalpha and retinoid X receptor alpha (RXRalpha). ANS binding is competed by PPARgamma agonists such as rosiglitazone, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), and 9,10-dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) (CAY10410). Moreover, the affinity of PPARgamma for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARgamma agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ(2) that does not activate PPAR in cells. We found that although this compound bound to PPARgamma with low affinity, it failed to promote PPARgamma interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARgamma binding and interactions may provide valuable strategies to fully understand the role of PPARgamma ligands.

Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA1: focus on vimentin.

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.