Guido Camici

Purification, Characterization, and Amino Acid Sequence of Cerato-platanin, a New Phytotoxic Protein from Ceratocystis fimbriata f. sp. platani

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp.platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S—S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank™) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus.Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.

PDGF receptor as a specific in vivo target for low M r phosphotyrosine protein phosphatase

Low M r phosphotyrosine protein phosphatase (LMWPTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW‐PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C12XXXXXR18, a main feature of all PTPase family members. In order to identify the LMW‐PTP substratels we have expressed in NIH‐3T3 cells a catalytically inert Cys12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW‐PTP expressing cells has enabled us to demonstrate an association between LMW‐PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW‐PTP on the phosphorylated platelet derived growth factor receptor.

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis.

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

Antidiabetic activity of some pentacyclic acid triterpenoids, role of PTP-1B: in vitro, in silico, and in vivo approaches.

The aim of the current study was to investigate the oral antidiabetic activity of four structurally-related triterpenic acids: ursolic (RE-01), oleanolic (RE-02), moronic (RE-03) and morolic (RE-04) acids. STZ-nicotinamide diabetic rats were treated with these triterpenes (50 mg/kg) and the antidiabetic effects in acute experiment were determined. All compounds showed significant antidiabetic activity in comparison with control group (p<0.05). The in vitro inhibitory activity of compounds against protein tyrosine phosphatase 1B (PTP-1B) was also evaluated. At 50 μM, the enzymatic activity was almost completely inhibited. All compounds were docked with a crystal structure of PTP-1B. Docking results suggested the potential binding of the triterpenic acids in a binding pocket next to the catalytic site. An extensive hydrogen bond network with the carboxyl group and Van der Waals interactions stabilize the protein-ligand complexes.

Protein N-homocysteinylation induces the formation of toxic amyloid-like protofibrils.

Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the epsilon-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia.

A new synthesis of benzoyl phosphate: a substrate for acyl phosphatase assay.

A new method for the synthesis of benzoyl phosphate was reported. The advantages are: 1. more rapid procedure; 2. lower cost; 3. higher yield.

Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes

Five phosphotyrosine‐containing peptides have been synthesized by FMOC solid‐phase peptide synthesis. These peptides correspond to the 411–419 sequence of the Xenopus src oncogene, to the 1191–1220 sequence of the human EGF receptor precursor, to the 1146–1158 sequence of the human insulin receptor, to the 856–865 sequence of the human, β‐PDGF receptor, and to the 5–16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter E1A plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyse all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.

THE MOLECULAR BASIS OF THE DIFFERING KINETIC BEHAVIOR OF THE TWO LOW MOLECULAR MASS PHOSPHOTYROSINE PROTEIN PHOSPHATASE ISOFORMS

The low molecular mass phosphotyrosine protein phosphatase is a cytosolic enzyme of 18 kDa. Mammalian species contain a single gene that codifies for two distinct isoenzymes; they are produced through alternative splicing and thus differ only in the sequence from residue 40 to residue 73. Isoenzymes differ also in substrate specificity and in the sensitivity to activity modulators. In our study, we mutated a number of residues included in the alternative 40-73 sequence by substituting the residues present in the type 2 isoenzyme with those present in type 1 and subsequently examined the kinetic properties of the purified mutated proteins. The results enabled us to identify the molecular site that determines the kinetic characteristics of each isoform; the residue in position 50 plays the main role in the determination of substrate specificity, while the residues in both positions 49 and 50 are involved in the strong activation of the type 2 low M phosphotyrosine protein phosphatase isoenzyme by purine compounds such as guanosine and cGMP. The sequence 49-50 is included in a loop whose N terminus is linked to the β2-strand and whose C terminus is linked to the α2-helix; this loop is very near the active site pocket. Our findings suggest that this loop is involved both in the regulation of the enzyme activity and in the determination of the substrate specificity of the two low M phosphotyrosine protein phosphatase isoenzymes.

Some protein tyrosine phosphatases target in part to lipid rafts and interact with caveolin-1

A profile-based search of the SWISS-PROT database reveals that most protein tyrosine phosphatases (PTPs) contain at least one caveolin-1-binding motif. To ascertain if the presence of caveolin-binding motif(s) in PTPs corresponds to their actual localization in caveolin-1-enriched membrane fractions, we performed subcellular fractionating experiments. We found that all tested PTPs (PTP1B, PTP1C, SHPTP2, PTEN, and LAR) are actually localized in caveolin-enriched membrane fractions, despite their distribution in other subcellular sites, too. More than 1/2 of LAR and about 1/4 of SHPTP2 and PTP-1C are localized in caveolin-enriched membrane fractions whereas, in these fractions, PTP-1B and PTEN are poorly concentrated. Co-immunoprecipitation experiments with antibodies specific for each tested PTP demonstrated that all five phosphatases form molecular complexes with caveolin-1 in vivo. Collectively, our findings propose that particular PTPs could perform some of their cellular actions or are regulated by recruitment into caveolin-enriched membrane fractions.

Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two lowMr phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowMr acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.