Prolactin/growth hormone-derived antiangiogenic peptides highlight a potential role of tilted peptides in angiogenesis

in Proceedings of the National Academy of Sciences of the United States of America (2006), 103(39), 14319-14324

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short ... [more ▼]

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's beta-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis. [less ▲]

Nanoscale Modification Of Supported Lipid Membranes: Synergetic Effect Of Phospholipase D And Viral Fusion Peptides

in Journal of Biomedical Nanotechnology (2005), 1(1), 1-8

Understanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various ... [more ▼]

Understanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. In this study, we used atomic force microscopy (AFM) to address the crucial question as to whether negatively curved lipids influence the ability of a viral fusion peptide to perturb the organization of supported lipid bilayers. To this end, an original approach was developed that makes use of an AFM tip functionalized with phospholipase D (PLD) enzymes to generate in situ small amounts of negatively curved phosphatidic acid (PA) in mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers. Real-time AFM imaging revealed that this nanomodification dramatically enhanced subsequent interaction with the simian immunodeficiency virus (SIV) fusion peptide. At short incubation time, the SIV peptide induced a 1.9 nm thickness reduction of the DPPC domains, reflecting either interdigitation or fluidification of the lipids. At longer incubation time, these depressed domains transformed into elevated striated domains, protruding one to several nanometers above the bilayer surface. Two complementary experiments, i.e. addition of the peptide onto DOPC/DPPC/DOPA bilayers or onto DOPC/DPPC bilayers pretreated with a PLD solution, confirmed that both PA and SIV peptides are required to induce depressed and striated domains. Accordingly, this is the first time that a high-resolution imaging technique is used to demonstrate that negatively curved lipids affect the membrane activity of fusion peptides. We believe the nanoscale approach presented here, i.e. use of enzyme-functionalized AFM tips to modify lipid bilayers, will find exciting new applications in nanobiotechnology for the design of biomimetic surfaces. [less ▲]

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity.

in Biochemical and Biophysical Research Communications (2005), 331(4), 894-901

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the ... [more ▼]

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling. [less ▲]

Determination Of The Functionality Of Common Apoa5 Polymorphisms

in Journal of Biological Chemistry (2005), 280(31), 28215-20

Common variants of APOA5 have consistently shown association with differences in plasma triglyceride (TG) levels. These single nucleotide polymorphisms (SNPs) fall into three common haplotypes: APOA5*1 ... [more ▼]

Common variants of APOA5 have consistently shown association with differences in plasma triglyceride (TG) levels. These single nucleotide polymorphisms (SNPs) fall into three common haplotypes: APOA5*1, with common alleles at all sites; APOA5*2, with rare alleles of -1131T--> C, -3A--> G, 751G--> T, and 1891T--> C; and APOA5*3, distinguished by the c56C--> G (S19W). Molecular modeling of the apoAV signal peptide (SP) showed an increased angle of insertion (65 degrees ) at the lipid/water interface of Trp-19 SP compared with Ser-19 SP (40 degrees ), predicting reduced translocation. This was confirmed by 50% reduction of Trp-19-encoded SP.secretory alkaline phosphatase (SEAP) fusion protein secreted into the medium from HepG2 cells compared with the Ser-19.SEAP fusion protein (p < 0.002). Considering APOA5*2 SNPs, there was no significant difference in the relative luciferase expression in Huh7 cells transiently transfected with a -1131T construct compared with the -1131C (fragments -1177 to -516 or -1177 to -3). Similarly, for the -3A--> G in the Kozak sequence, in vitro transcription/translation assays and primer extension inhibition assays showed no alternate AUG initiation codon usage, demonstrating that -3A--> G did not influence translation efficiency. Although 1891T--> C in the 3'-untranslated region disrupts a putative Oct-1 transcription factor binding site, when inserted 3' of the luciferase gene the T--> C change demonstrated no significant difference in luciferase expression. Thus, association of APOA5*2 SNPs with TG levels is not due to the individual effects of any of these SNPs, although cooperativity between the SNPs cannot be excluded. Alternatively, the effect on TG levels may reflect the strong linkage disequilibrium with the functional APOC3 SNPs. [less ▲]

Transferrin-Binding Protein B Of Neisseria Meningitidis: Sequence-Based Identification Of The Transferrin-Binding Site Confirmed By Site-Directed Mutagenesis

in Journal of Bacteriology (2004), 186(3), 850-7

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of ... [more ▼]

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains. [less ▲]

Structural investigation of reconstituted high density lipoproteins by scanning tunnelling microscopy

in Applied Surface Science (2004), 203

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have ... [more ▼]

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have a detailed knowledge of the structure and molecular organisation of HDL. Reconstituted nascent high density lipoproteins (r-HDL), consisting of synthetic phospholipids together with different apolipoproteins (apo A-I, A-IV and E), were thus analysed by scanning tunnelling microscopy (STM). Both shape and dimensions of the discoidal HDL particles measured by this technique were found in good agreement with the data available from the literature. The accuracy of the STM pictures presented in this paper enables for the first time the visualisation of the molecular organisation of such macromolecules. The arrangement of the protein as antiparallel helical segments, is consistent with the general mode of organisation of apolipoprotein/phospholipid discoidal particles previously reported. [less ▲]

Piracetam Inhibits The Lipid-Destabilising Effect Of The Amyloid Peptide A Beta C-Terminal Fragment

in Biochimica et Biophysica Acta-Biomembranes (2003), 1609(1), 28-38

Amyloid peptide (Abeta) is a 40/42-residue proteolytic fragment of a precursor protein (APP), implicated in the pathogenesis of Alzheimer's disease. The hypothesis that interactions between Abeta ... [more ▼]

Amyloid peptide (Abeta) is a 40/42-residue proteolytic fragment of a precursor protein (APP), implicated in the pathogenesis of Alzheimer's disease. The hypothesis that interactions between Abeta aggregates and neuronal membranes play an important role in toxicity has gained some acceptance. Previously, we showed that the C-terminal domain (e.g. amino acids 29-42) of Abeta induces membrane permeabilisation and fusion, an effect which is related to the appearance of non-bilayer structures. Conformational studies showed that this peptide has properties similar to those of the fusion peptide of viral proteins i.e. a tilted penetration into membranes. Since piracetam interacts with lipids and has beneficial effects on several symptoms of Alzheimer's disease, we investigated in model membranes the ability of piracetam to hinder the destabilising effect of the Abeta 29-42 peptide. Using fluorescence studies and 31P and 2H NMR spectroscopy, we have shown that piracetam was able to significantly decrease the fusogenic and destabilising effect of Abeta 29-42, in a concentration-dependent manner. While the peptide induced lipid disorganisation and subsequent negative curvature at the membrane-water interface, the conformational analysis showed that piracetam, when preincubated with lipids, coats the phospholipid headgroups. Calculations suggest that this prevents appearance of the peptide-induced curvature. In addition, insertion of molecules with an inverted cone shape, like piracetam, into the outer membrane leaflet should make the formation of such structures energetically less favourable and therefore decrease the likelihood of membrane fusion. [less ▲]

Analysis Of Accessible Surface Of Residues In Proteins

in Protein Science : A Publication of the Protein Society (2003), 12(7),

Analysis Of The C-Terminal Membrane Anchor Domains Of Hepatitis C Virus Glycoproteins E1 And E2: Toward A Topological Model

in Journal of Virology (2002), 76(4), 1944-58

The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen ... [more ▼]

The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen where they remain. The 31 C-terminal residues of the E1 protein and the 29 C-terminal residues of the E2 protein are implicated in the ER retention. Moreover, the E1 and E2 C termini are implicated in E1-E2 heterodimerization. We studied the E1 and E2 C-terminal sequences of 25 HCV strains in silico using molecular modeling techniques. We conclude that both C-terminal domains should adopt a similar and peculiar configuration: one amphipathic alpha-helix followed by a pair of transmembrane beta-strands. Several three-dimensional (3-D) models were generated. After energy minimization, their ability to interact with membranes was studied using the molecular hydrophobicity potentials calculation and the IMPALA procedure. The latter simulates interactions with a membrane by a Monte Carlo minimization of energy. These methods suggest that the beta-hairpins could anchor the glycoproteins in the ER membrane at least transiently. Anchoring could be stabilized by the adsorption of the nearby amphipathic alpha-helices at the membrane surface. The 3-D models correlate with experimental results which indicate that the E1-E2 transmembrane domains are involved in the heterodimerization and have ER retention properties. [less ▲]

Autosomal recessive segregation of a truncating mutation of anti-Mullerian type II receptor in a family affected by the persistent Mullerian duct syndrome contrasts with its dominant negative activity in vitro.

in Journal of Clinical Endocrinology and Metabolism (2001), 86(9), 4390-7

Anti-Mullerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Mullerian hormone type II ... [more ▼]

Anti-Mullerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Mullerian hormone type II receptor occur naturally, causing the persistent Mullerian duct syndrome. In a family with two members with persistent Mullerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Mullerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Mullerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Mullerian duct syndrome. Truncating mutations in receptors of the TGFbeta family exert dominant negative activity, which was seen only when each of the mutant anti-Mullerian hormone receptors was overexpressed in an anti-Mullerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis. [less ▲]