Corinne Rommens

The complete amino acid sequence of bovine milk angiogenin

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas‐phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14 577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His‐14, Lys‐41 and His‐115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg‐Gly‐Asp which is not present in the human protein.

Direct evidence of cytoplasmic delivery of PKC-α, -ϵ and -ζ pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC‐α, ‐ϵ, and ‐ζ pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti‐apoptotic program.

Fenofibrate Inhibits Endothelin-1 Expression by Peroxisome Proliferator–Activated Receptor α–Dependent and Independent Mechanisms in Human Endothelial Cells

Objective—Dyslipidemia contributes to endothelial dysfunction in type 2 diabetes mellitus. Fenofibrate (FF), a ligand of the peroxisome proliferator–activated receptor-&agr; (PPAR&agr;), has beneficial effects on microvascular complications. FF may act on the endothelium by regulating vasoactive factors, including endothelin-1 (ET-1). In vitro, FF decreases ET-1 expression in human microvascular endothelial cells. We investigated the molecular mechanisms involved in the effect of FF treatment on plasma levels of ET-1 in type 2 diabetes mellitus patients. Methods and Results—FF impaired the capacity of transforming growth factor-&bgr; to induce ET-1 gene expression. PPAR&agr; activation by FF increased expression of the transcriptional repressor Krüppel-like factor 11 and its binding to the ET-1 gene promoter. Knockdown of Krüppel-like factor 11 expression potentiated basal and transforming growth factor-&bgr;–stimulated ET-1 expression, suggesting that Krüppel-like factor 11 downregulates ET-1 expression. FF, in a PPAR&agr;-independent manner, and insulin enhanced glycogen synthase kinase-3&bgr; phosphorylation thus reducing glycogen synthase kinase-3 activity that contributes to the FF-mediated reduction of ET-1 gene expression. In type 2 diabetes mellitus, improvement of flow-mediated dilatation of the brachial artery by FF was associated with a decrease in plasma ET-1. Conclusion—FF decreases ET-1 expression by a PPAR&agr;-dependent mechanism, via transcriptional induction of the Krüppel-like factor 11 repressor and by PPAR&agr;-independent actions via inhibition of glycogen synthase kinase-3 activity.

Glucose Regulates the Expression of the Apolipoprotein A5 Gene

The apolipoprotein A5 gene (APOA5) is a key player in determining triglyceride concentrations in humans and mice. Since diabetes is often associated with hypertriglyceridemia, this study explores whether APOA5 gene expression is regulated by alteration in glucose homeostasis and the related pathways. d-Glucose activates APOA5 gene expression in a time- and dose-dependent manner in hepatocytes, and the glycolytic pathway involved was determined using d-glucose analogues and metabolites. Together, transient transfections, electrophoretic mobility shift assays and chromatin immunoprecipitation assays show that this regulation occurs at the transcriptional level through an increase of USF1/2 binding to an E-box in the APOA5 promoter. We show that this phenomenon is not due to an increase of mRNA or protein expression levels of USF. Using protein phosphatases 1 and 2A inhibitor, we demonstrate that d-glucose regulates the APOA5 gene via a dephosphorylation mechanism, resulting in an enhanced USF1/2-promoter binding. Last, subsequent suppressions of USF1/2 and phosphatases mRNA through siRNA gene silencing abolished the regulation. We demonstrate that the APOA5 gene is up regulated by d-glucose and USF through phosphatase activation. These findings may provide a new cross-talk between glucose and lipid metabolism.

Chemoselective acylation of hydrazinopeptides: a novel and mild method for the derivatization of peptides with sensitive fatty acids

N-terminal α-hydrazinoacetylpeptides were synthesized and chemoselectively acylated on the hydrazine moiety with various fatty acid succinimidyl esters or N-(cholesterylcarbonyloxy) succinimide. The acylation was performed in water/2-methyl-propane-2-ol mixtures buffered at pH 5.1. The mild reaction conditions allow the derivatization of peptides by sensitive fatty acids.

Generation and characterization of a humanized PPARδ mouse model

BACKGROUND AND PURPOSE Humanized mice for the nuclear receptor peroxisome proliferator‐activated receptor δ (PPARδ), termed PPARδ knock‐in (PPARδ KI) mice, were generated for the investigation of functional differences between mouse and human PPARδ and as tools for early drug efficacy assessment.

A novel and mild solid phase hydroperoxydeamination reaction

Abstract The unnatural amino acid Lys(NH2), easily incorporated into a peptidyl-PEGA resin by N-electrophilic amination of the e-amino group, was cleanly transformed on the solid phase into 6-hydroxynorleucine by air oxidation in the presence of bicarbonate ions followed by reduction of the hydroperoxy intermediate with a water soluble phosphine.

Apolipoprotein A-V Modulates Insulin Secretion in Pancreatic β-cells Through its Interaction with Midkine

Apolipoprotein A-V is an important determinant of plasma triglyceride level in both humans and mice. This study showed the physiological impact of apoA-V on insulin secretion in rat pancreatic β-cells (INS-1 cells). In order to precise the mechanism of action, binding experiments coupled to mass spectrometry were performed to identify a potential membrane receptor. Results showed an interaction between apoA-V and midkine protein. Confocal microscopy confirmed the plasma membrane co-localisation of this two-proteins after the treatment of INS-1 cells with the apo-AV recombinant protein and indicated that the cell surface midkine could be involved in apoA-V endocytosis, since these two proteins were co-translocated at the plasma membrane or in the cytosol compartment. This co-localisation is correlated with an increase in insulin secretion in a dose dependant manner during short incubation period. Reduction of midkine expression by small interfering RNA duplexes revealed a decrease in the ability of these transfected cells to secrete insulin in presence of apoA-V. Competition experiments for the apoA-V-midkine binding at the cell surface using antibody directed against midkine is able to influence INS-1 cell function as insulin secretion. Our results showed apoA-V ability to enhance insulin secretion in β-cells and provide evidence of an internalization pathway involving the midkine as partner.

Chemistry of hydrazinopeptides: a new hydroperoxydeamination process

Abstract The N -alkyl hydrazino group of hydrazinopeptides 1 is oxidized by air in bicarbonate buffers into the corresponding hydroperoxy moiety in high yield.

Convergent synthesis of D-(–)-quinic and shikimic acid-containing dendrimers as potential C-lectin ligands by sulfide ligation of unprotected fragments

The preparation of D-(–)-quinic and (–)-shikimic acid-derived dendrimers with valencies of 4, 8 and 16, respectively, as potential C-lectin ligands is reported. D-(–)-Quinic and shikimic acids were branched to an (S-tert-butylthio-L-cysteine)-containing tripeptide on solid phase to furnish compounds 1 and 3. These intermediates were reduced upon treatment with tri-n-butylphosphine and linked to N-chloroacetylated L-lysinyl cores via a nucleophilic substitution performed in aqueous DMF.