Luciana Dente

The SH3 Domains of Endophilin and Amphiphysin Bind to the Proline-rich Region of Synaptojanin 1 at Distinct Sites That Display an Unconventional Binding Specificity

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the “classical” type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

pEMBL: a new family of single-stranded plasmids for sequencing DNA

Publisher Summary This chapter describes a series of plasmids, the “pEMBL family,” that have the property of being encapsidated as single-stranded deoxyribonucleic acid (DNA) upon superinfection with phage F1. The plasmids can be used for DNA sequencing and for all the other uses that require single-stranded DNA, but, in the absence of F1 helper phage, they replicate as any other doubles-stranded plasmid. This is very useful for the preparation of a large quantity of plasmid and for the stability of longer inserts. The class of M13 derivative vectors, largely used for DNA sequencing with the dideoxy method, has, in fact, the limitation that large inserts tend to be unstable. The chapter discusses the principle of the method and characteristics of pEMBL vectors along with the construction of pEMBL plasmids. The chapter discusses the property of a short segment of F1 genome containing all the cis-acting elements for DNA replication and morphogenesis. To have a series of polylinkers containing a large number of unique sites for restriction endonucleases, in different reciprocal orientation is very useful, because it allows the insertion of DNA fragments in a preselected orientation or in a preselected frame inside the sequence encoding the a-fragment of th/3-galactosidase. The DNA, which is prepared through the procedures described in the chapter, is suitable for DNA sequencing experiments with the dideoxy method.

Alternative Bacteriophage Display Systems

Filamentous phage has been extensively used to implement various aspects of phage display technology. The success of these organisms as vectors to present foreign peptides and to link them to their coding sequences is a consequence of their structural and biological characteristics. Some of these properties, however, represent a limitation when one attempts to display proteins that cannot be efficiently exported through the bacterial membrane or do not fold properly in the periplasm. Thus, the desirability of developing alternative display systems was recognised recently and led to the development of a different class of display vectors that assemble their capsid in the cytoplasm and are released via cell lysis. This review describes and compares the properties of these alternative display systems.

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex

The carboxy‐terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein–protein interaction. These domains (WW, EF hands, ZZ and H1–H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin‐associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy‐terminus. We found that none of the carboxy‐terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage‐displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in β‐dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW‐mediated binding to PPxY peptides and to β‐dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin β‐dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca2+‐dependent binding of calmodulin. The remaining two motifs found in the carboxy‐terminus (H1–H2) mediate the formation of utrophin–dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.

Anti-synapsin monoclonal antibodies: epitope mapping and inhibitory effects on phosphorylation and Grb2 binding

The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 (SH3) domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I (mAbs 10.22, 19.51, 19.11 and 19.8) and G for synapsin II (mAb 19.8). The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca2+/calmodulin-dependent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I.

Domain repertoires as a tool to derive protein recognition rules.

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.

Non-mammalian vertebrate embryos as models in nanomedicine

UNLABELLED Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems. Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues and antagonism from the anti-vivisectionist movement. Recently, there has been increasing interest in the scientific community in the interactions between nano-materials and non-mammalian developmental organisms, which are now being recognized as valid models for the study of human disease. This review examines and discusses the biomedical applications and the interaction of nano-materials with embryonic systems, focusing on non-mammalian vertebrate models, such as chicken, zebrafish and Xenopus. FROM THE CLINICAL EDITOR Animal models are critical components of preclinical biomedical research. This review discusses the feasibility and potential applications of non-mammalian vertebral animals, such as zebrafish, xenopus, and chicken as animal models in nanomedicine research.

The orientation of the neuronal growth process can be directed via magnetic nanoparticles under an applied magnetic field

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields (Ms). The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. In a neuron-like cell line, we have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction. From the clinical editor: This study validates that non-invasive approaches for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields are possible. The hypothesis was confirmed by observing preferential neurite outgrowth in a cell culture system along the direction imposed by an external magnetic field.

Magnetic nanoparticles as intraocular drug delivery system to target Retinal Pigmented Epithelium (RPE)

One of the most challenging efforts in drug delivery is the targeting of the eye. The eye structure and barriers render this organ poorly permeable to drugs. Quite recently the entrance of nanoscience in ocular drug delivery has improved the penetration and half-life of drugs, especially in the anterior eye chamber, while targeting the posterior chamber is still an open issue. The retina and the retinal pigment epithelium/choroid tissues, located in the posterior eye chamber, are responsible for the majority of blindness both in childhood and adulthood. In the present study, we used magnetic nanoparticles (MNPs) as a nanotool for ocular drug delivery that is capable of specific localization in the retinal pigmented epithelium (RPE) layer. We demonstrate that, following intraocular injection in Xenopus embryos, MNPs localize specifically in RPE where they are retained for several days. The specificity of the localization did not depend on particle size and surface properties of the MNPs used in this work. Moreover, through similar experiments in zebrafish, we demonstrated that the targeting of RPE by the nanoparticles is not specific for the Xenopus species.