Monica Dettin

The Proprotein Convertase SKI-1/S1P IN VITRO ANALYSIS OF LASSA VIRUS GLYCOPROTEIN-DERIVED SUBSTRATES AND EX VIVO VALIDATION OF IRREVERSIBLE PEPTIDE INHIBITORS

Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations ≤100 μm these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases.

Synthetic peptides from the principal neutralizing domain of human immunodeficiency virus type 1 (HIV-1) enhance HIV-1 infection through a CD4-dependent mechanism

The principal neutralizing domain (PND) of Human Immunodeficiency Virus type 1 (HIV-1) is mapped to a 24-amino acid sequence located in the hypervariable V3 region of the viral envelope protein. The PND of HIV-1 isolates from infected individuals corresponds mostly to that of the HIV-1 MN strain. We found that a peptide designed from the PND of HIV-1 MN virus greatly enhanced viral infection, while a peptide-derived PND of HTLV-IIIB virus showed at least 10-fold less efficient activity; no such effect was exhibited by the other peptides tested, including one designed from the PND of HIV-1 RF strain. The observed enhancing effect occurred in the early steps of viral infection and was not strain-restricted as both MN- and IIIB-derived peptides increased heterologous virus expression, including that of the RF strain. The MN- and, to a lesser extent, IIIB-derived peptides also increased CD4 expression on the cell membrane and differentially inhibited CD4 down-regulation induced by the phorbol ester TPA and/or by the monosialoganglioside GM1; the peptides showing no viral infection enhancement had no such effects. These findings demonstrate that the viral enhancement observed took place through a CD4-dependent mechanism and suggest that the PND is involved in HIV-1 infection and spread.

Covalent surface modification of titanium oxide with different adhesive peptides: surface characterization and osteoblast-like cell adhesion.

A fundamental goal in the field of implantology is the design of innovative devices suitable for promoting implant-to-tissue integration. This result can be achieved by means of surface modifications aimed at optimizing tissue regeneration. In the framework of oral and orthopedic implantology, surface modifications concern both the optimization of titanium/titanium alloy surface roughness and the attachment of biochemical factors able to guide cellular adhesion and/or growth. This article focuses on the covalent attachment of two different adhesive peptides to rough titanium disks. The capability of biomimetic surfaces to increase osteoblast adhesion and the specificity of their biological activity due to the presence of cell adhesion signal-motif have also been investigated. In addition, surface analyses by profilometry, X-ray photoelectron spectroscopy, and time of flight-secondary ion mass spectrometry have been carried out to investigate the effects and modifications induced by grafting procedures.

Electrospun scaffolds of self-assembling peptides with poly(ethylene oxide) for bone tissue engineering

Structural, mechanical and biochemical properties have to be considered when searching for suitable extracellular matrix substitutes. Fibrous structures of synthetic or natural polymers have received increasing interest as three-dimensional scaffolds for tissue engineering applications as they can be easily produced by electrospinning with different topographical features by changing the process parameters. On the other hand, the nanobiotechnology approach suggests mimicking molecular architectures in nature through self-assembly. In particular, self-assembling peptide-based biomaterials have been successfully used as scaffolds for cell growth. In order to amalgamate these two strategies nanofibrous electrospun scaffolds of hybrid polymer were designed and obtained by mixing poly(ethylene oxide) and self-assembling peptides in aqueous solution. The results of in vitro osteoblast adhesion and proliferation assays on the electrospun scaffolds obtained using different self-assembling peptide sequences are discussed.

Assessment of novel chemical strategies for covalent attachment of adhesive peptides to rough titanium surfaces: XPS analysis and biological evaluation

Bioactive molecules have been proposed to promote beneficial interactions at bone-implant interfaces for enhancing integration. The main objective of this study was to develop novel methods to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive peptides, through selective reaction involving single functional groups. In the first protocol, an aminoalkylsilane was covalently linked to the Ti oxide layer, followed by covalent binding of glutaric anhydride to the free NH(2) groups. The carboxylic group of glutaric anhydride was used to condense the free N-terminal group of the side-chain protected peptide sequence. Finally, the surface was treated with trifluoroacetic acid to deprotect side-chain groups. In the second protocol, the peptide was directly anchored to the Ti oxide surface via UV activation of an arylazide peptide analogue. X-ray photoelectron spectroscopy analyses confirmed that modifications induced onto surface composition were in agreement with the reactions performed. The peptide density of each biomimetic surface was determined on the basis of radiolabeling and XPS derived reaction yields. The in vitro cellular response of the biomimetic surfaces was evaluated using a primary human osteoblast cell model. Cell adhesion, proliferation, differentiation, and mineralization were examined at initial-, short-, and long-time periods. In was shown that the biomimetic surface obtained through photoprobe-marked analogue that combines an easily-performed modification provides a favorable surface for an enhanced cellular response.

Self-assembling peptide-enriched electrospun polycaprolactone scaffolds promote the h-osteoblast adhesion and modulate differentiation-associated gene expression

Electrospun polycaprolactone (PCL) is able to support the adhesion and growth of h-osteoblasts and to delay their degradation rate to a greater extent with respect to other polyesters. The drawbacks linked to its employment in regenerative medicine arise from its hydrophobic nature and the lack of biochemical signals linked to it. This work reports on the attempt to add five different self-assembling (SA) peptides to PCL solutions before electrospinning. The hybrid scaffolds obtained had regular fibers (SEM analysis) whose diameters were similar to those of the extracellular matrix, more stable hydrophilic (contact angle measurement) surfaces, and an amorphous phase constrained by peptides (DSC analysis). They appeared to have a notable capacity to promote the h-osteoblast adhesion and differentiation process by increasing the gene expression of alkaline phosphatase, bone sialoprotein, and osteopontin. Adding an Arg-Gly-Asp (RGD) motif to a self-assembling sequence was found to enhance cell adhesion, while the same motif condensed with a scrambled sequence did not, indicating that there is a cooperative effect between RGD and 3D architecture created by the self-assembling peptides. The study demonstrates that self-assembling peptide scaffolds are still able to promote beneficial effects on h-osteoblasts even after they have been included in electrospun polycaprolactone. The possibility of linking biochemical messages to self-assembling peptides could lead the way to a 3D decoration of fibrous scaffolds.

BINDING TO CD4 OF SYNTHETIC PEPTIDES PATTERNED ON THE PRINCIPAL NEUTRALIZING DOMAIN OF THE HIV-1 ENVELOPE PROTEIN

The interaction between the viral envelope protein gp120 and the cellular surface antigen CD4 is a key event in HIV-1 infection. Reciprocal high affinity binding sites have been located in the first domain of CD4 and in the carboxy-terminal region of gp120, respectively. Upon infection, the membranes of the target cells fuse; sites of CD4 and gp120, distinct from their high affinity binding sites, play a role in the post-binding events leading to syncytia formation. We have studied the interactions of CD4 with gp120 and gp120-derived peptides using an in vitro assay based on immobilized recombinant soluble CD4 (sCD4). In this system CD4 binds to recombinant soluble gp120 and to anti-receptor peptides derived from the high affinity CD4-binding site of gp120, as well as to peptides corresponding to the principal neutralizing domain (PND) of the envelope protein, i.e., to the domain required for HIV-1-mediated syncytium formation. Competition experiments performed using epitope-specific mAbs and a variety of peptides indicated that PND-derived peptides are specifically recognized by a CD4 site adjacent to, but distinct from, the high affinity gp120-binding site of CD4. Synthetic peptides patterned on the PND of different viral isolates were retained onto sCD4-based affinity columns at different extent; some of the structural requirements for binding were analyzed. Studies performed on CD4+ T-cells showed that PND-derived peptides also interact with CD4 in its native membrane-bound conformation. These results indicate that a direct contact takes place between CD4 and the gp120 domain participating in HIV-induced syncytia formation.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)4K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)4K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)4K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)4K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright

Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.

Chemoselective Surface Immobilization of Proteins through a Cleavable Peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.