Anna Caselli

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.

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.

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.

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.

PrPc activation induces neurite outgrowth and differentiation in PC12 cells: role for caveolin-1 in the signal transduction pathway

Cellular prion protein (PrPc) is a ubiquitous glycoprotein, whose physiological role is poorly characterized. It has been suggested that PrPc participates in neuritogenesis, neuroprotection, copper metabolism, and signal transduction. In this study we detailed the intracellular events induced by PrPc antibody‐mediated cross‐linking in PC12 cells. We found a Fyn‐dependent activation of the Ras‐Raf pathway, which leads to a rapid and transient phosphorylation of extracellular regulated kinases. In addition, this activation cascade relies on the engagement of integrins, and involves focal adhesion kinase activation. We demonstrated the tyrosine phosphorylation of caveolin‐1 as a consequence of PrPc stimulation, and showed that phosphocaveolin‐1 scaffolds and coordinates protein complexes involved in PrPc‐dependent signaling. Moreover, we found that caveolin‐1 phosphorylation, is a mechanism for recruiting the C‐terminal Src kinase and inactivating Fyn, so as to terminate cell signaling. Furthermore our data support a significant role for PrPc as a response mediator in neuritogenesis and cell differentiation.

In vivo inactivation of phosphotyrosine protein phosphatases by nitric oxide.

The effect of NO on phosphotyrosine protein phosphatases (PTPases) has been investigated in vivo. NO production is induced in interferon‐γ and lipopolyaccharide stimulated RAW‐264.7 macrophages as indicated by the increase of NO2 − in the medium. Our results demonstrate an inhibition of p‐nitrophenylphosphatase activity as a consequence of macrophages activation. Under the described experimental conditions, most of the hydrolysis of p‐nitrophenylphosphate can be ascribed to the action of cellular PTPases. The presence of , a specific inhibitor of NO synthase decreases the inactivation rate of both membrane‐bound and soluble PTPases. This evidence further confirms the ability of NO to inactivate PTPases and suggests a possible role of NO in the regulation of cellular processes involving this class of phosphatases.

The insulin-mimetic effect of Morin: A promising molecule in diabetes treatment

BACKGROUND Type-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs. METHODS Selection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [(3)H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program. RESULTS We demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a Ki in the μM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth. CONCLUSIONS We have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only. GENERAL SIGNIFICANCE Our study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.

Synthesis, biological activity and structure–activity relationships of new benzoic acid-based protein tyrosine phosphatase inhibitors endowed with insulinomimetic effects in mouse C2C12 skeletal muscle cells

Insulin resistance is a complex altered metabolic condition characterized by impaired insulin signaling and implicated in the pathogenesis of serious human diseases, such as diabetes, obesity, neurodegenerative pathologies. In pursuing our aim to identify new agents able to improve cellular insulin sensitivity, we have synthesized new 4-[(5-arylidene-4-oxo-2-phenylimino/oxothiazolidin-3-yl)methyl]benzoic acids (5, 8) and evaluated their inhibitory activity towards human protein tyrosine phosphatases PTP1B, LMW-PTP and TCPTP, enzymes which are involved in the development of insulin resistance. Compounds 5 and 8 showed from moderate to significant selectivity toward PTP1B over both the highly homologous TCPTP and the two isoforms of human LMW-PTP. In addition, most of the tested compounds selectively inhibited LMW-PTP IF1 over the isoform IF2. Docking studies into the active sites of PTP1B and LMW-PTP aided the rationalization of the observed PTP inhibitory profile. Moreover, most tested compounds were capable to induce the insulin metabolic pathway in mouse C2C12 skeletal muscle cells by remarkably stimulating both IRβ phosphorylation and 2-deoxyglucose cellular uptake.

5-Fluorouracil resistant colon cancer cells are addicted to OXPHOS to survive and enhance stem-like traits

Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.