Monica Salamone

The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.

Dipeptidyl peptidase IV (DPP4/CD26) and seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.

An active form of sphingosine kinase-1 is released in the extracellular medium as component of membrane vesicles shed by two human tumor cell lines.

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

Differential expression and function of cadherin-like proteins in the sea urchin embryo

Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.

A New Method to Value Efficiency of Enzyme Blends for Pancreatic Tissue Digestion

Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, >50% of islet procedures do not eventuate in transplantation due both to the variability of a donors pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with vertebrate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, breakdown, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets.

Intracellular trafficking of endogenous fibroblast growth factor-2.

We have previously reported how the release of fibroblast growth factor‐2 (FGF‐2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF‐2 is targeted to the budding vesicles. Considering that in vitro cultured Sk‐Hep1 hepatocarcinoma cells release FGF‐2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF‐2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in FGF‐2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF‐2 granules moving to the cell periphery and FM4‐64‐labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF‐2 release, were analyzed for their effects on FGF‐2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF‐2 movements toward the cell periphery. The FGF‐2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF‐2 clustered at the cell periphery, but the rate of its release decreased. FGF‐2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF‐2 translocation to the nucleus and nucleolus. FGF‐2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms.

Comprehensive Analysis of a Vibrio parahaemolyticus Strain Extracellular Serine Protease VpSP37.

Proteases play an important role in the field of tissue dissociation combined with regenerative medicine. During the years new sources of proteolytic enzymes have been studied including proteases from different marine organisms both eukaryotic and prokaryotic. Herein we have purified a secreted component of an isolate of Vibrio parahaemolyticus, with electrophoretic mobilities corresponding to 36 kDa, belonging to the serine proteases family. Sequencing of the N-terminus enabled the in silico identification of the whole primary structure consisting of 345 amino acid residues with a calculated molecular mass of 37.4 KDa. The purified enzyme, named VpSP37, contains a Serine protease domain between residues 35 and 276 and a canonical Trypsin/Chimotrypsin 3D structure. Functional assays were performed to evaluate protease activity of purified enzyme. Additionally the performance of VpSP37 was evaluated in tissue dissociations experiments and the use of such enzyme as a component of enzyme blend for tissue dissociation procedures is strongly recommended.

Proteolytic enzymes clustered in specialized plasma-membrane domains drive endothelial cells' migration

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

Development of a fast DNA extraction method for sea food and marine species identification

The authentication of food components is one of the key issues in food safety. Similarly taxonomy, population and conservation genetics as well as food web structure analysis, also rely on genetic analyses including the DNA barcoding technology. In this scenario we developed a fast DNA extraction method without any purification step from fresh and processed seafood, suitable for any PCR analysis. The protocol allows the fast DNA amplification from any sample, including fresh, stored and processed seafood and from any waste of industrial fish processing, independently of the sample storage method. Therefore, this procedure is particularly suitable for the fast processing of samples and to carry out investigations for the authentication of seafood by means of DNA analysis.

Transcriptional and Biochemical Effects of Cadmium and Manganese on the Defense System of Octopus vulgaris Paralarvae

Due to anthropogenic activities the relative concentrations of cadmium and manganese have increased in the marine environment. Cephalopods are able to accumulate such metals and, as inhabitant of coastal waters, Octopus vulgaris is continuously exposed to anthropogenic activities. Since no study is available on the effects of heavy metals at molecular level in developing octopuses, herein we exposed 1-day-old paralarvae for 24 h to 10, 100, and 1000 μg/L of CdCl2 or MnCl2. Cd exerted a concentration-dependent inhibition of survival and a reduction in growth rate was shown while Mn exposure did not affect the survival rate even at the highest concentrations. Gene expression profiles of hsp70, sod, cat, and gst genes were analyzed by quantitative real-time PCR and defined patterns of transcription were observed. Moreover posttranscriptional analyses were also performed suggesting the impairment of metabolic functions, under strong oxidative conditions (as occurred in paralarvae exposed to Cd) or the complete detoxification events (as occurred in paralarvae exposed to Mn).

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations.