Riccardo Marzocchini

Differential role of four cysteines on the activity of a low M r phosphotyrosine protein phosphatase

In this paper we describe the construction or five mutants of a bovine liver low M r phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide‐directed mutagenesis Cys‐17, Cys‐62 and Cys‐145 were converted to Ser while Cys‐12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p‐nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein or E. coli; both of the Cys‐12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys‐17 mutant was greatly decreased (200‐fold). The Cys‐62 mutant showed a 2.5‐fold decrease in specific activity, while the Cys‐145 mutant remained almost unchanged. These data confirm the involvement of Cys‐12 and Cys‐17 in the catalytic site and suggest that Cys‐62 and Cys‐145 mutations may destabilise the structure of the enzyme.

A proteomic approach to identify plasma proteins in patients with abdominal aortic aneurysm.

Our aim was to identify the key proteins involved in the pathogenesis of AAAs. To explore the possible pathogenetic mechanisms involved in AAA, we analyzed by proteomics modifications in plasma proteome of patients with AAA. Therefore, the present study analyzed the soluble plasma proteins using two dimensional electrophoresis (2-DE) and mass spectrometry (MS). We identified 33 protein spots, 31 of which show an up-regulation in AAA patients whilst the expression level of 2 protein spots is reduced. We confirm a number of biomarkers associated with AAA that have been previously identified by various authors. We identified a significant increase of a class of proteins such as fibrinogen, α1-antitrypsin and haptoglobin in plasma from AAA patients. The presence of these proteins in human AAA plasma may be related to the inflammatory processes active in these subjects. We have seen a negative correlation between the vitamin D-binding protein (DBP) and hemoglobin subunit β and AAA presence. DBP levels have been found to increase in AAA wall tissues by others and this discrepancy with our results could be due to the different analysis source. We wanted to analyze the factors measurable in plasma-associated rather than in tissue-associated markers because the application of circulating biomarkers in diagnostic laboratories would be relatively simple. DBP is very important for vascular remodelling and it may have an important role in the protection of vascular walls. In plasma tissue this protein reduces platelet aggregation and extends coagulation time. No one protein identified in this study has the biologic plausibility to be used singularly as a biomarker of aneurysmal disease due to inadequate specificity. The effect of using multiple biomarkers combined with clinical factors requires investigation in carefully designed population-based studies and these studies need to select the criteria of choice to define healthy controls very carefully. Clearer identification of various markers is needed, possibly using other proteomic techniques to screen for new candidates such as gel-free proteomic technology that enables us to handle larger groups of subject compared to gel-based proteomic technology.

The expression of low molecular weight protein tyrosine phosphatase is up‐regulated in 1,2‐dimethylhydrazine‐induced colon tumours in rats

Recent studies have assessed the role of low molecular weight protein tyrosine phosphatase (LMW‐PTP) in cell transformation and tumour onset and progression, observing a significant increase in the expression of LMW‐PTP mRNA and protein in human breast, colon, bladder and kidney tumour samples. Moreover, its enhanced expression is generally prognostic of a more aggressive cancer. To better understand the role of this protein during colon carcinogenesis and to study whether its overexpression is also observed in earlier phases of carcinogenesis, we studied its expression in colon tumours, induced in rats by treatment with 1,2‐dimethylhydrazine (DMH), an animal model that resemble the sequential formation of histopathological lesions of spontaneous carcinogenesis in humans. The results show a significant increase in LMW‐PTP expression in adenocarcinomas, suggesting that this phenomenon is associated with the onset of malignancy. Moreover a significant overexpression of LMW‐PTP transcript is associated with tumours originating in the proximal (right) part of the colon, confirming an observation already reported for human colon cancer.

The 5′‐untranslated region of the human muscle acylphosphatase mRNA has an inhibitory effect on protein expression

The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5′‐untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem‐loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10‐fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5′‐untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.

Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1

The genes of the human low M r phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S‐transferase. The recombinant proteins were purified by a rapid one‐step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X‐ray‐grade, diffracted to better than 2.4 Å and were suitable for high resolution X‐ray structure determination.

Cloning and expression of the cDNA coding for the erythrocyte isoenzyme of human acylphosphatase

Three independent cDNAs coding for the erythrocyte isoform of human acylphosphatase were isolated and characterized. All the clones were incomplete at the 5′ end, but Northern blot analysis using the cDNA as a probe showed the presence of an unusually long mRNA 5′‐untranslated region. The transcript was present in a variety of human cell lines of different origins, although at different levels. Southern blot analysis on DNA from different individuals revealed a simple hybridization pattern. Large amounts of pure enzyme with kinetic characteristics very similar to those of the native protein were expressed in E. coli.

Cloning, expression and characterisation of a new human low Mr phosphotyrosine protein phosphatase originating by alternative splicing

RT‐PCR experiments on RNA from K562 and HepG2 cells and from human placenta led to the isolation of a novel cDNA, a further alternative splicing product of the primary transcript of low M r phosphotyrosine phosphatase (LMW‐PTP), already known to produce isoforms 1 and 2. This new transcript represents 15–20% of the total LMW‐PTP mRNA in the cell. This novel cDNA codifies for a protein that we have named SV3 (splicing variant 3): the deduced protein sequence presents the first 49 residues identical to those of isoform 1, followed by 24 unrelated amino acids, due to a frameshift introduced at the novel exon‐exon boundary. The SV3 protein, expressed in E. coli is enzymatically inactive, most probably because unfolded, as suggested by far‐UV circular dichroism (CD) experiments. SV3 protein appears to possess the characteristics of an unstructured polypeptide chain lacking the packing of side chain residues and the secondary structure level that are typical of globular proteins. This protein could represent an inactive variant of the human LMW‐PTP.

Acylphosphatase expression during macrophage differentiation and activation of U-937 cell line

The two acylphosphatase isoenzymes (muscle type and common type) are differently involved in cell differentiation processes. In this paper we investigate the expression of the two isoenzymes during macrophage differentiation and activation. The U-937 human promonocytic cell line is a model for cell differentiation induced by the tumor promoter phorbol myristic acetate (PMA). Here we show that only the expression of the muscle type acylphosphatase increases during U-937 differentiation and macrophage activation, confirming that the two isoenzymes are differently regulated. Moreover, we determined, in the same conditions, the level of specific mRNA. Results show that after an initial two-fold decrease during PMA stimulation, the muscle type acylphosphatase mRNA levels remain constant also after the treatment with lipopolysaccharide and gamma-interferon, treatments that lead to macrophage activation. It is possible that post-transcription regulation is responsible for the regulation of muscle type acylphosphatase in the cell during differentiation and macrophage activation.

Characterization of a novel Drosophila melanogaster acylphosphatase

Analysis of the Drosophila melanogaster EST database led to the characterization of a novel acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher K cat but lower K m with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes.

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet‐derived growth factor

Low M r phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet‐derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X‐100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low M r PTP overexpression.