Biomedical subjects

L Castagnoli

Publications and source records attributed to L Castagnoli.

At least 19 recordsLinked to original sources

PubMed2005

Handgrip impairment in Charcot-Marie-Tooth disease.

AIM: Charcot-Marie-Tooth disease (CMT) is a genetic neuropathy causing muscle weakening in the feet, legs and hands, with consequent impairment of ambulation and handgrip. For fast clinical evaluation and rehabilitation management of handgrip deficits, a functional classification in 4 stages or levels of clinical severity, based on the loss of handgrip types from the finest to the roughest, has been recently proposed. The aim of this study is to evaluate the prevalence of each level of handgrip impairment in a wide population of patients affected with demyelinating and axonal CMT. METHODS: Two-hundred and forty-eight non-operated hands were examined to evaluate if and how the pinch between the pulp of the thumb and the pulp of the second or third finger was made, starting from the palm-up position with the fingers abducted or, in case of impossibility to do so, if a lateral pinch or only a grasp was possible. Following to this observation, each hand was fitted in 1 of the 4 stages described in the above-mentioned classification and then the frequency of each stage was determined. RESULTS: As a whole, 75.4% hands were at stage 1; 9.7 were at stage 2; 10.9% at stage 3; 4% at stage 4. CONCLUSIONS: The results of this survey reveal that, in the majority of the CMT cases, handgrip is affected mildly so that only simple recommendations to prevent further muscle and joint damage are required; however, in more than 1 out 5 cases, the handrip impairment is quite severe and requires a detailed rehabilitative program with daily exercises, and, in a small number of cases, is so severe that independence in the daily living activities is lost or very reduced.

Adolescent↗

PubMed2001

Selection of ligands by panning of domain libraries displayed on phage lambda reveals new potential partners of synaptojanin 1.

One of the goals of functional genomics is the description of reliable and complete protein interaction networks. To facilitate ligand discovery from complex protein mixtures, we have developed an improved approach that is affected by a negligible fraction of false positives. We have combined a novel technique based on the display of cDNA libraries on the capsid of bacteriophage lambda and an efficient plaque assay to reveal phage displaying ligands that are enriched after only a couple of affinity purification steps. We show that the lambda display system has a unique ability to display, at high density, proteins ranging in size from a few to at least 300 amino acid residues. This characteristic permits attenuation of the size bias in the selection procedure and, at the same time, offers a sensitive plaque assay that permits us to do away with the ligand background without unduly increasing the number of selection cycles. By using a proline-rich fragment of the synaptojanin 1 protein as a bait, we have identified, in a brain cDNA display library, seven ligands all containing either SH3 or WW domains. Four of these correspond to proteins that have already been validated as physiological partners, while the remaining three are new partners, whose physiological relevance remains to be established. Two different proline-rich regions of the p21-activated protein kinase 1 (Pak1) and WAVE/SCAR2 protein retrieve from the library different proteins containing SH3 or WW domains.

Amino Acid Sequence↗

PubMed2001

Physical interaction with Yes-associated protein enhances p73 transcriptional activity.

Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. Here we report that the nuclear phosphoprotein p73 can engage in a physical association with the Yes-associated protein (YAP). This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine (Y) of the PPPPY motif of p73 was shown to be essential for the association with YAP. Unlike p73alpha, p73beta, and p63alpha, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73gamma isoform do not interact with YAP. Indeed, we documented that YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73alpha transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2001

A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli.

The development of a convenient and promising alternative to the various two-hybrid methods that are used to study protein-protein interactions is described. In this system, a lambdoid chimeric operator is recognized by a hybrid repressor formed by two chimeric monomers whose C-terminal domains are composed of heterologous proteins (or protein domains). Only if these proteins efficiently dimerize in vivo is a functional repressor formed able to bind the chimeric operator and shut off the synthesis of a downstream reporter gene. This new approach was tested with several interacting proteins ranging in size from less than 100 to more than 800 amino acids and, to date, no size or topology limit has been detected.

Bacteriophages↗

PubMed2001

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.

Bacteriophages↗

PubMed2001

Carl von Rokitansky and the Italian translation of the Handbuch der Pathologischen Anatomie: a linguistic and doctrinal enigma.

Carl von Rokitansky was the author of a treatise that came out between 1842 and 1846 with the title Handbuch der Pathologischen Anatomie. A historical milestone in pathological anatomy, Rokitansky's work represented the first attempt to systematically classify pathological specimens. Its publication inevitably made a great impact on Vienna, at that time the major European medical centre. The Italian translation of Rokitansky's masterpiece, Trattato Completo di Anatomia Patologica, published in Venice in 1852, was carried out by Ricchetti and Fano: the former a philologist and the latter a Triestine physician who, in 1873, had worked at Simon Pertot's side, the first prosector to be assumed in Trieste. From the start, the two translators not only made no secret of the linguistic obstacles they came up against, but also how unconvincing Rokitansky's doctrines were; a scepticism emerged from their words that inevitably contributed to the realization of a translation difficult to read. Undoubtedly, Rokitansky elaborated a theory of disease containing a certain degree of unclarity and in this respect it is interesting to emphasize that even the English translation, Manual of Pathologic Anatomy (1849-1854), demonstrated similar conceptual problems. A convinced supporter of gross pathology, Rokitansky put forth a theory of disease, the so-called Krasenlehre, resting upon humoral doctrines. This new knowledge inevitably exerted a great influence over Viennese, as well as German, medicine. Rokitansky's humoral pathology survived until the 1850s, when it was attacked by young Virchow, the future, universally recognized, father of cellular pathology, who definitively extinguished speculative humoral pathology.

Anatomy↗

PubMed2000

Physical and functional interaction between p53 mutants and different isoforms of p73.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Breast Neoplasms↗

PubMed2000

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.

Amino Acid Motifs↗

PubMed1999

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.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Phage displayed peptide libraries.

Peptide libraries may be constructed by grafting, in vitro, random DNA sequences into a carrier gene and then introducing the degenerate hybrid coding sequence into an expression organism. This review will focus on phage display, which was the first expression organism for peptide library expression to be described and which still maintains predominance in this area because of its simplicity, minimal cost, ease of manipulation, power and robustness. Using phage as the host, a repertoire of random peptides can be expressed that may be searched by a variety of screening or selection procedures. By physically associating each element of the peptide library with its coding sequence, selection for a property of a specific peptide results in the enrichment of the corresponding gene thus facilitating its cloning and amplification. This review focuses on the construction and screening of peptide libraries displayed on filamentous phage capsid and only briefly discusses the display of proteins and protein domains.

Animals↗

PubMed1998

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Recognition specificity of individual EH domains of mammals and yeast.

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.

Adaptor Proteins, Signal Transducing↗

PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome VII.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Base Sequence↗

PubMed1997

The genes encoding the transcription factor yTAFII60, the G4p1 protein and a putative glucose transporter are contained in a 12.3 kb DNA fragment on the left arm of Saccharomyces cerevisiae chromosome VII.

We report the nucleotide sequence of a DNA fragment of 12,325 base pairs from the left arm of the Saccharomyces cerevisiae chromosome VII. Inspection of the coding capacity revealed 11 open reading frames (ORFs) longer than 100 amino acids. Five ORFs are significantly homologous to known proteins. The region encoding ORF G2985 corresponds (100%) to the gene encoding the yeast TATA binding protein-associated factor TAFII60. The G3075 ORF is 47.8% identical to the hypothetical yeast protein YB88. G3089 shows 36.7% identity to the eel calmodulin. G3085 shows 94.9% identity with the published sequence of the quadruplex DNA binding protein G4p1. G3090 reveals 46.7% identity with the probable glucose transport protein yBR1625.

Amino Acid Sequence↗

PubMed1997

Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.

Amino Acid Sequence↗

PubMed1995

A novel approach to protein-protein interaction: complex formation between the p53 tumor suppressor and the HIV Tat proteins.

By using a novel genetic approach, based on the properties of lambda cl repressor, we demonstrate that the HIV-1 Tat protein specifically interacts with the human p53 protein via the p53 O2 dimerization domain. By random and site-specific mutagenesis, we also identify the residues in Tat and O2 peptides which are involved in this interaction. Two alternative biological consequences are expected to result from Tat-p53 interaction: (i) Tat-O2 interaction inactivates p53 regulation function, thus producing cell transformation; (ii) Tat-O2 interaction favours the formation of p53 dimers, thus leading the cell towards apoptosis.

Amino Acid Sequence↗

PubMed1994

Linking an easily detectable phenotype to the folding of a common structural motif. Selection of rare turn mutations that prevent the folding of Rop.

Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.

Amino Acid Sequence↗

PubMed1993

Increasing the immunogenicity of protein antigens through the genetic insertion of VQGEESNDK sequence of human IL-1 beta into their sequence.

The immunogenicity of two recombinant protein Ag containing the immunostimulatory sequence of human IL-1 beta 163-171 (VQGEESNDK) genetically engineered into their structure has been evaluated. The IL-1 beta sequence was inserted into the loop between alpha helices D and E of recombinant human ferritin H chain and into the hypervariable region of recombinant flagellin from Salmonella muenchen. The chimeric proteins were injected into mice and the level of humoral immune response developed against the native proteins was assessed by measuring the number of Ag-specific plaque forming cells/spleen or as the level of serum IgG response. The response was compared to that of mice receiving injections with wild-type protein Ag not containing the VQGEESNDK sequence or with hybrid constructs containing unrelated foreign peptide sequences of the same length. A significantly higher immune response was observed in mice immunized with chimeric constructs containing the human IL-1 beta 163-171 sequence. These data suggest that the insertion of the VQGEESNDK sequence may prove useful to increase the immune response against poorly immunogenic recombinant proteins.

Adjuvants, Immunologic↗