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S Alberti

Publications and source records attributed to S Alberti.

At least 37 records · Page 2Linked to original sources
PubMed1999

Evolution of the genetic code, protein synthesis and nucleic acid replication.

A better definition of the structural and thermodynamic determinants of the interaction of nucleic acids with proteins is shedding light on the origin of the genetic code, protein synthesis, and nucleic acid replication. This is also allowing to show a consistent biochemical framework for the appearance of these fundamental synthetic mechanisms. This article reviews recent significant developments in the field, and discusses an integrated model for a biochemically plausible evolution of these fundamental mechanisms of synthesis. This model is based on sequence-specific interactions between abiotically synthesized polynucleotides and polypeptides, and can account for a coordinate evolution of the genetic code, protein synthesis, and nucleic acid replication in living cells.

DNA↗
PubMed1999

Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex.

ADP-Ribosylation Factors↗
PubMed1999

HIKE, a candidate protein binding site for PH domains, is a major regulatory region of Gbeta proteins.

HIKE is a highly conserved sequence motif that selectively occurs in proteins candidate to bind PH domains, e.g., the beta subunit of heterotrimeric G proteins, kinases, ankyrin and kinesin. Thus, the HIKE region has been predicted to be a protein docking site for PH domains. This work evidentiates recent experimental evidence that unambiguously defines the functional role of HIKE in Gbeta as a multiple effector docking site and as a major regulatory region of G protein's function. Indeed, the Gbeta HIKE interacts with the beta-adrenergic receptor kinase, Galpha, Ggamma, adenylyl cyclase 2, phospholipase C beta2, inward rectifier K channels, calcium channel alpha1B, calmodulin, phosducin, ste20. Quite interestingly, HIKE is located in the Gbeta region that faces the cell membrane. Thus, HIKE also interacts with the cell membrane and may dynamically regulate membrane vs effector binding of the Galphabetagamma trimer. These findings fulfill a major prediction of the HIKE model, i.e., that HIKE is a regulatory region for protein-protein interactions. A role of HIKE as a proteic binding site for PH domains is supported by the profound influence of HIKE mutations on the largely PH-mediated binding of beta-ARK to Gbeta.

Amino Acid Sequence↗
PubMed1999

Identification of a novel DNA binding site for nuclear orphan receptor OR1.

The nuclear orphan receptor OR1 has been shown to bind as a heterodimer with retinoid X receptor (RXR) to direct repeat 4 (DR4) response elements. It remained unclear, however, whether this represents the only or the optimal binding site for this receptor. Therefore, we performed a DNA binding site selection assay that allows the identification of novel DNA binding sites for OR1 in an unbiased manner. While OR1 alone was not able to select a specific sequence from the pool of oligonucleotides, the OR1/RXR heterodimer selected a highly conserved DR1 element, termed DR1s, with two AGGTCA motifs spaced by one adenosine. The functional activity of the consensus binding site was verified in transient transfection assays and corroborated by in vitro studies. Based on the sequence of the consensus DR1s, we located putative natural binding sites in the 5'-promoter flanking regions of the rat S14 gene and the rat cholecystokinin type A receptor gene. Furthermore, we could show that although the OR1/RXR heterodimer has a distinct binding orientation on a DR4 element, it is able to bind in both orientations to the DR1s element. The OR1 paralog LXRalpha does not bind as a heterodimer with RXR to the DR1s element, indicating that these receptors, despite their homology, are involved in the regulation of different sets of genes.

Animals↗
PubMed1999

Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniae clinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O- strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.

Denmark↗
PubMed1998

Human Trop-2 is a tumor-associated calcium signal transducer.

Trop-2/EGP-1/GA733-1 is a recently identified cell surface glycoprotein highly expressed by human carcinomas. The cytoplasmic tail of Trop-2 possesses potential serine and tyrosine phosphorylation sites and a phosphatidyl-inositol binding consensus sequence. Thus, we investigated whether Trop-2 might be a functional signaling molecule. Using the fluorescent probe Fura-2, we assayed the cytoplasmic calcium levels in human cancer cells stimulated with anti-Trop-2 or control antibodies. Three anti-Trop-2 MAbs, Rs7-7G11, MOv16 and 162-46.2 specifically induced a transient intracellular calcium level increment in up to 40% of the experiments performed. Polyclonal antisera recognizing recombinant Trop-2 molecules possessed a much lower stimulation efficiency. The average latency of antibody-induced Ca2+ rise for OvCa-432 cells was 64+/-26 sec. Internal Ca2+ concentrations reached peaks of 190+/-24 nM vs. basal levels of 61+/-4 nM and returned to baseline within 193+/-37 sec. Similar values were obtained in MCF-7 cells. For comparison, stimulation of P2-purinergic receptors on MCF-7 and OvCa-432 cells induced a Ca2+ rise in most cases, leading to average internal Ca2+ concentrations of 297+/-41 and 391+/-39 nM, respectively. Our findings show that Trop-2 transduces an intracellular calcium signal, are consistent with the hypothesis that it acts as a cell surface receptor and support a search for a physiological ligand.

Adenosine Triphosphate↗
PubMed1998

A phosphoinositide-binding sequence is shared by PH domain target molecules--a model for the binding of PH domains to proteins.

Pleckstrin homology (PH) domains have been proven to bind phosphoinositides (PI) and inositolphosphates (IP). On the other hand, a binding of PH domains to proteins is still a matter of debate. The goal of this work was to identify potential PH domain protein target sites and to build a model for PH domain-protein binding. A candidate sequence, called HIKE, was identified by sequence homology analysis of the proteins that are considered the strongest PH binding candidates, i.e., Gbeta, PKC, and Akt. HIKE contains a PI binding sequence and fulfills several criteria for a potential PH-binding site, i.e., it is present in other PH-binding candidates, lies in regulatory regions independently predicted to bind PH domains, and is conserved in 3-D structure among different molecules. These findings and the similarities with the mode of binding of PTB and PDZ domains suggest a beta strand-beta strand coordination model for PH-protein binding. The HIKE model predicts that membrane anchoring of PH domains and their targets could be a critical step in their interaction, which would consistently explain why PH-protein binding has only been detected in the presence of PI.

Amino Acid Sequence↗
PubMed1998

Cloning of the murine TROP2 gene: conservation of a PIP2-binding sequence in the cytoplasmic domain of TROP-2.

Trop-2 is a novel calcium signal transducer expressed at high levels by most human carcinomas. To develop an animal model to study the function of this molecule in vivo, we have cloned the murine Trop2 gene. Using human TROP2 primers, we amplified by PCR a segment of murine Trop2. This was used as a probe to clone a full-length gene by hybridization of a genomic library. The cloned murine Trop2 gene is functional, as indicated by sequencing and by expression after transfection. The murine Trop2 is 87.4% similar to its human homologue, with the highest conservation in the extracellular region between residues 86 and 157. Essentially all cysteines are conserved between the human and the murine genes, suggesting conservation of the Trop2 disulfide bridges and of its overall structure. Intriguingly, the cytoplasmic tail of Trop2 shows a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sequence, which overlaps with a protein kinase C phosphorylation site. Thus, we speculate that PIP2 might regulate the phosphorylation state of Trop2 and play a role in its signal transduction. Murine Trop2 mRNA is detected in normal kidney, lung, ovary and testis, similarly to the human gene. Interestingly, the highest levels of expression are found in immortalized keratinocytes. Since Trop2 is undetectable in undifferentiated spindle cell carcinomas, this suggests a preferential expression at early stages of tumor progression.

Amino Acid Sequence↗
PubMed1997

The origin of the genetic code and protein synthesis.

A model for a parallel evolution of the genetic code and protein synthesis is presented. The main tenet of this model is that the genetic code, that is, a correspondence between nucleotide and amino-acid coding units, originated from sequence-specific interaction between abiotically synthesized polynucleotides and polypeptides. A sequence-specific binding between oligonucleotides and oligopeptides is supported by experimental findings. Moreover, it is parsimonious enough to be consistent with the relatively simple chemistry of a primordial environment. Proximity between peptides and RNA increased the rate of formation of ester bonds between them. This lead to the accumulation of sequence-specific polypeptide-polynucleotide pairs, that is, of primordial-loaded tRNA. Condensation of short polypeptides into longer products could be catalyzed by a sequence-specific juxtaposition of loaded tRNA over complementary RNA, originating the core of protein synthesis. The accumulation of useful encoded products, for example, catalysts for tRNA loading (primordial aminoacyl-tRNA synthetases) or stabilizers of tRNA-mRNA interactions (primordial ribosomes), permitted the subsequent evolution of protein synthesis and of the genetic code to their mature form. This occurred via a parallel reduction in length of the interacting polynucleotides and polypeptides. Thus, it maintained the correct reading frame of mRNA from the preceding stages of evolution.

Evolution, Molecular↗
PubMed1997

Detection of the receptor for the human urokinase-type plasminogen activator using fluoresceinated uPA.

The urokinase-type plasminogen activator (uPA) is a serine protease that plays a crucial role in blood coagulation and in tumor invasion and metastasis. uPA is a relatively large polypeptide and binds the uPA receptor (uPAR) with high affinity and specificity. Therefore, it was a good candidate for direct labeling with a fluorochrome for detection of the uPAR. We have produced a fluorescein (FITC)-labeled human uPA using a conjugation procedure that did not significantly alter its binding characteristics to the uPAR. Thirty nM FITC-uPA efficiently stains 2 x 10(5) uPAR-transfected mouse cells in suspension, as determined by flow cytometric analysis. One microgram of FITC-uPA efficiently stains 2 x 10(5) uPAR transfectants grown on slides and analyzed by fluorescence optical microscopy. Human cell lines expressing the endogenous uPAR were stained with similar efficiency. Fixation in paraformaldehyde only slightly reduced the efficiency of staining of both transfectants and cell lines. These characteristics allow the use of FITC-uPA in both static and dynamic morphological studies of uPAR-expressing cells.

Affinity Labels↗
PubMed1996

A high affinity T cell receptor?

T cell receptors (TCR) have a low affinity for the MHC and the presented peptides (MHCpep). The low affinity of the TCR is crucial in T cell recognition and activation. Nevertheless, I propose that the TCR is perfectly capable of specifically binding MHCpep with high affinity. This hypothesis is supported by several data. Among them, the frequency of negatively selected immature T cells and of high affinity alloreactive T cells. Structural and functional analysis indicates that the TCR binding regions are quite similar to those of immunoglobulins. This implies similar binding strategies and suggests that there are no structural constraints on TCR affinity. The possible existence of high affinity TCR is relevant for current views on immune recognition, alloreactivity and peptide antagonism. In particular, it supports a model of immune recognition as unselected higher affinity binding to newly encountered peptides. The actual production of specific affinity TCR may also prove crucial for a soluble T cell receptor-based immunotherapy and for the co-crystallization of TCR with MHC and peptides.

Animals↗
PubMed1996

Expression of HLA class I genes in meiotic and post-meiotic human spermatogenic cells.

In human spermatogenic cells, in contrast to somatic cells, expression of major histocompatibility complex (MHC) class I molecules is undetectable. This lack of expression may contribute to the absence of female immune reaction against spermatozoa and may be necessary for gamete fusion. Among the molecular repressor mechanisms that may be used at the DNA level, we investigated 5' CpG methylation of the different class Ia and class Ib loci in meiotic pachytene spermatocytes and postmeiotic round spermatids, which had been purified from human testes by centrifugal elutriation. These results were compared with those obtained with mature spermatozoa and peripheral blood mononuclear cells. Using methylation-sensitive restriction enzymes and DNA locus-specific probes, we found that HLA-A, HLA-B/C, and HLA-E loci were similarly unmethylated in the germ and somatic cells tested, whereas HLA-F and HLA-G were even less methylated in the former cells. Together with the observation that spermatozoon DNA contains class I genes that are transfectable and able to direct transcription and protein synthesis in murine L cells, these data suggest that HLA class I genes are in an active conformation in male germ cells. We indeed found that both spermatocytes and spermatids contained low levels of class Ia and class Ib mRNA. Using reverse transcriptase-polymerase chain reaction, followed by DNA sequencing, we also detected three HLA-G transcriptional isoforms, resulting from alternative splicings, which suggested that this class Ib gene may have a potential function in these germ cells. Although intracellular expression of beta2-microglobulin (the light chain that associates with HLA class I heavy chains) was found in spermatocytes but not in round spermatids, no membrane-bound nor intracellular translated HLA class I heavy chain was detected in either germ cell type, when monomorphic anti-HLA class I monoclonal antibodies were used. Thus, lack of expression of HLA class I proteins in the male germ line is likely to involve post-transcriptional mechanisms of regulation.

Alternative Splicing↗