GnRH receptor expression in human prostate cancer cells is affected by hormones and growth factors.

GnRH receptors (GnRH-R) have been found in various malignancies, including prostate cancer (PCa). They mediate the direct antitumor effects of GnRH analogs. Nevertheless, few reports concern drug-induced modulation of GnRH-R levels. In this study, we investigated GnRH-R expression in androgen-sensitive (LNCaP) and -insensitive (PC-3) PCa cells treated for 4 and 6 days with a GnRH agonist (Leuprorelin acetate, LA, 10(-11) or 10(-6) M), Dihydrotestosterone (DHT, 10(-9) M), Cyproterone acetate (CA, 10(-7) M), and Epidermal growth factor (EGF, 10 ng/ml), either alone or combined. The RT-PCR analysis showed no variation in GnRH-R mRNA levels of both treated LNCaP and PC-3 cells. On the contrary, immunoblotting indicated that in LNCaP and PC-3 cells, LA upregulated membrane GnRH-R expression (up to 92%). In androgen-sensitive cells, DHT induced a GnRH-R increase (up to 119%) always comparable to that occurring in the presence of CA. GnRH-R upregulation by LA/DHT or CA/DHT association was similar to that promoted by the single agents. In PC-3 cells, EGF upregulated GnRH-R (up to 110%). A prolonged treatment (for 12 days) determined a greater EGF-induced increase in GnRH-R levels (142%). Lower (or no) receptor enhancement occurred when LA and EGF were associated. Our findings indicate that LA post-transcriptionally upregulates its own membrane receptor in androgen-sensitive and -insensitive PCa cells, counteracting the receptor enhancement produced by DHT and EGF. The effects, obtained with a relatively long and continuous treatment, may have implications in the choice of therapy modality with GnRH analogs and may render the receptor a novel therapeutic target, particularly in hormone-refractory PCa.

Genetic risk factors in typical haemolytic uraemic syndrome.

BACKGROUND: Haemolytic uraemic syndrome (HUS) is a disorder characterized by thrombotic microangiopathy, which is caused in 'typical forms' by gastrointestinal infections with Escherichia Coli species that produce verotoxins. Several studies have identified negative prognostic factors of the disease, among which prolonged oliguria, neurological involvement and increased leukocytosis have been more consistently reported. We have hypothesized that the genetic background may also predispose to the development of typical forms of HUS and may influence the clinical course of the disease. METHODS: Fourteen polymorphisms, known to influence the coagulation pathway or the activity of the renin-angiotensin system, have been selected and studied in 150 Italian children with typical forms of HUS. Two hundred healthy Italian children were used as controls. RESULTS: The risk of developing HUS was strongly associated with the platelet glycoprotein 1balpha 145M allele (OR 3.08; CI: 1.62-5.85) (P < 0.001). A significant association was also found with polymorphisms located in the adipocyte-derived leucine aminopeptidase and factor V genes. A longer duration of dialysis was moderately associated with increased leukocytosis and with the 807T allele of the platelet glycoprotein 1a gene. High white blood cell count was also strongly associated with the risk of long-term sequelae (OR 2.91, CI: 1.21-6.98) (P < 0.02), whereas the 1166C allele of the angiotensin II type 1 receptor had a significant protective effect (OR 0.28, CI: 0.09-0.83) (P < 0.02). CONCLUSIONS: These results highlight the role of glycoprotein 1balpha in the physiopathology of typical forms of HUS and show that the genetic background plays a role in the susceptibility and severity of the disease.

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates.

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to epsilon-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.

Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines.

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules.

Peptide recognition modules mediate many protein-protein interactions critical for the assembly of macromolecular complexes. Complete genome sequences have revealed thousands of these domains, requiring improved methods for identifying their physiologically relevant binding partners. We have developed a strategy combining computational prediction of interactions from phage-display ligand consensus sequences with large-scale two-hybrid physical interaction tests. Application to yeast SH3 domains generated a phage-display network containing 394 interactions among 206 proteins and a two-hybrid network containing 233 interactions among 145 proteins. Graph theoretic analysis identified 59 highly likely interactions common to both networks. Las17 (Bee1), a member of the Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins, showed multiple SH3 interactions, many of which were confirmed in vivo by coimmunoprecipitation.

Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: structural and functional analysis.

Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-A resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.