Bridged bicyclic peptides as potential drug scaffolds: synthesis, structure, protein binding and stability.

Double cyclization of short linear peptides obtained by solid phase peptide synthesis was used to prepare bridged bicyclic peptides (BBPs) corresponding to the topology of bridged bicyclic alkanes such as norbornane. Diastereomeric norbornapeptides were investigated by 1H-NMR, X-ray crystallography and CD spectroscopy and found to represent rigid globular scaffolds stabilized by intramolecular backbone hydrogen bonds with scaffold geometries determined by the chirality of amino acid residues and sharing structural features of ß-turns and α-helices. Proteome profiling by capture compound mass spectrometry (CCMS) led to the discovery of the norbornapeptide 27c binding selectively to calmodulin as an example of a BBP protein binder. This and other BBPs showed high stability towards proteolytic degradation in serum.

Crosstalk between inflammation, iron metabolism and endothelial function in Behçet's disease.

Behçet's disease (BD) is a rare chronic vasculitis of unclear etiology. It has been suggested that inflammatory response has an important role in BD pathophysiology. Herein, we aimed to study the interplay between inflammation, iron metabolism and endothelial function in BD and search for its putative association with disease activity. Twenty five patients clinically diagnosed with BD were selected and twenty four healthy age-sex matched individuals participated as controls. Results showed an increase of total number of circulating white blood cells and neutrophils, serum transferrin, total iron binding capacity, mieloperoxidase (MPO), ceruloplasmin (Cp), C reactive protein, ß2 microglobulin and Cp surface expression in peripheral blood monocytes in BD patients comparatively to healthy individuals (p < 0,05). Of notice, the alterations observed were associated to disease activity status. No significant differences between the two groups were found in serum nitric oxide concentration. The results obtained suggest an important contribution from innate immunity in the pathogenesis of this disease. In particular, surface expression of leukocyte-derived Cp may constitute a new and relevant biomarker to understand BD etiology.

Immune cells and hepatocytes express glycosylphosphatidylinositol-anchored ceruloplasmin at their cell surface.

BACKGROUND: Ceruloplasmin is a positive acute-phase protein with both anti- and pro-oxidant activities, thus having still unclear physiological functions in inflammatory processes. Importantly, ceruloplasmin has been implicated in iron metabolism due to its ferroxidase activity, assisting ferroportin on cellular iron efflux. Ceruloplasmin can be expressed as a secreted or as a membrane glycosylphosphatidylinositol-anchored protein (GPI-ceruloplasmin), this latter one being reported as expressed mostly in the brain. DESIGN AND METHODS: We studied the expression of both ceruloplasmin isoforms in human peripheral blood lymphocytes, monocytes, mouse macrophages and human hepatocarcinoma cell line HepG2, using immunofluorescence and immunoblotting techniques. Co-localization of ceruloplasmin and ferroportin was also investigated by immunofluorescence in mouse macrophages. RESULTS: Ceruloplasmin was detected by immunoblotting and immunofluorescence in membrane and cytosol of all cell types. The cell surface ceruloplasmin was identified as the GPI-isoform and localized in lipid rafts from monocytes, macrophages and HepG2 cells. In macrophages, increased expression levels and co-localization of ferroportin and GPI-ceruloplasmin in cell surface lipid rafts were observed after iron treatment. Such iron upregulation of ceruloplasmin was not observed in HepG2. CONCLUSIONS: Our results revealed an unexpected ubiquitous expression of the GPI-ceruloplasmin isoform in immune and hepatic cells. Different patterns of regulation of ceruloplasmin in these cells may reflect distinct physiologic functions of this oxidase. In macrophages, GPI-ceruloplasmin and ferroportin likely interact in lipid rafts to export iron from cells. Precise knowledge about ceruloplasmin isoforms expression and function in various cell types will help to clarify the role of ceruloplasmin in many diseases related to iron metabolism, inflammation and oxidative biology.

Diagnostic and prognostic biomarker discovery strategies for autoimmune disorders.

Current clinical, laboratory or radiological parameters cannot accurately diagnose or predict disease outcomes in a range of autoimmune disorders. Biomarkers which can diagnose at an earlier time point, predict outcome or help guide therapeutic strategies in autoimmune diseases could improve clinical management of this broad group of debilitating disorders. Additionally, there is a growing need for a deeper understanding of multi-factorial autoimmune disorders. Proteomic platforms offering a multiplex approach are more likely to reflect the complexity of autoimmune disease processes. Findings from proteomic based studies of three distinct autoimmune diseases are presented and strategies compared. It is the authors' view that such approaches are likely to be fruitful in the movement of autoimmune disease treatment away from reactive decisions and towards a preventative stand point.

Ceruloplasmin expression by human peripheral blood lymphocytes: a new link between immunity and iron metabolism.

Ceruloplasmin (CP) is a multicopper oxidase involved in the acute phase reaction to stress. Although the physiological role of CP is uncertain, its role in iron (Fe) homeostasis and protection against free radical-initiated cell injury has been widely documented. Previous studies showed the existence of two molecular isoforms of CP: secreted CP (sCP) and a membrane glycosylphosphatidylinositol (GPI)-anchored form of CP (GPI-CP). sCP is produced mainly by the liver and is abundant in human serum whereas GPI-CP is expressed in mammalian astrocytes, rat leptomeningeal cells, and Sertolli cells. Herein, we show using RT-PCR that human peripheral blood lymphocytes (huPBL) constitutively express the transcripts for both CP molecular isoforms previously reported. Also, expression of CP in huPBL is demonstrated by immunofluorescence with confocal microscopy and flow cytometry analysis using cells isolated from healthy blood donors with normal Fe status. Importantly, the results obtained show that natural killer cells have a significantly higher CP expression compared to all other major lymphocyte subsets. In this context, the involvement of lymphocyte-derived CP on host defense processes via its anti/prooxidant properties is proposed, giving further support for a close functional interaction between the immune system and the Fe metabolism.

Lymphocyte ceruloplasmin and Behçet's disease.

INTRODUCTION: Behçet's disease (BD) is a rare chronic inflammatory disorder of unknown aetiology. However, it has been postulated that a dysregulation of the prooxidant/antioxidant balance may be important to its pathogenesis. Ceruloplasmin (CP) is an acute phase protein expressed at the surface of peripheral blood lymphocytes (PBL) with antioxidant properties and with a relevant role in iron (Fe) metabolism. OBJECTIVES: To study CP expression at the surface of PBL (PBLCP) in patients with BD. MATERIAL AND METHODS: We measured serum CP and PBLCP obtained from BD patients (n=10) and respective controls (n=10) using nephelometry and flow cytometry techniques, respectively. Additionally, haematological parameters, biochemical Fe metabolism markers [serum Fe, serum ferritin, serum transferrin, total Fe binding capacity (TIBC), transferrin saturation] and non-specific markers of inflammation [serum C reactive protein (CRP), beta2-microglobulin] were measured in all individuals. RESULTS: Despite the absence of significant differences between the two study groups when comparing serum CP, a significant difference in PBLCP was found in BD patients mainly due to a significant decrease of CP expression at the surface of CD3-CD56+ lymphocytes. Also, a significant decrease of PBLCP was observed in patients treated with azathioprine compared to patients that were not being treated with this drug. CONCLUSIONS: According to this study, we suggest that the significant decrease of PBLCP observed in BD patients might be due to azathioprine treatment and not directly related to the pathophysiology of BD.