Analyses of human endogenous retrovirus-encoded proteins with potential relevance for human biology and diseases

The human genome harbors numerous sequences derived from ancient retroviral infections, so-called human endogenous retroviruses (HERVs). The HERVs of the HERV-K(HML-2) group, in short HML- 2, encode various retroviral proteins that, if expressed, may affect cell biology. HML-2 transcription is upregulated in different disease contexts including presence of HML-2 encoded proteins. Some HML-2 proteins have been implicated in disease development, although definitive associations with diseases were not reported yet. This work focused on HML-2 protease (Pro) and HML-2 integrase (IN), two HML-2 encoded proteins that received little attention so far, although their enzymatic activities can, in principle, have detrimental effects in the cell. In the first part of this work we investigated HML-2 Pro. Besides the retroviral proteins that constitute the virion, some retroviral aspartyl proteases, among them HIV-1 protease, are known to process cellular proteins. We wondered whether HML-2 Pro proteolytic activity can process cellular proteins as well. By cleaving cellular proteins, HML-2 Pro may impair cellular processes, thus have a role in disease development. The major aim for this part was to identify cellular proteins processed by HML- 2 Pro. We also performed initial experiments documenting HML-2 Pro activity against cellular proteins in a cellular environment and provided insights into cellular processes that could be potentially influenced by HML-2 Pro activity. HML-2 Pro was successfully purified and reaction conditions for assaying its activity in vitro were optimized. Purified HML-2 Pro was incubated with cellular proteins derived from HeLa cells. Employing terminal amine isotope labeling of substrates (TAILS) we then identified cellular proteins cleaved by HML-2 Pro. TAILS experiment and subsequent analyses of raw data produced a list of 872 human proteins as putative cellular substrates of HML-2 Pro. Identified proteins were profiled using suited bioinformatic tools. Processed proteins could be assigned to different cellular compartments and various, often disease-relevant cellular processes. We verified through additional experiments processing of selected candidate proteins by HML-2 Pro in vitro and in vivo. Cleavage by HML-2 Pro was confirmed for 9 out of 14 selected candidate proteins in vitro. Further verification experiments demonstrated in vivo cleavage of cellular proteins by HML-2 Pro in a cellular environment. Sizes of processing products observed for some of the tested proteins coincided with product sizes predicted by TAILS, thus corroborating TAILS results. We documented cell death and activation of apoptotic processes during HML-2 Pro overexpression, hence providing initial experimental evidence of cellular processes being influenced by HML-2 Pro activity. Finally, we obtained preliminary evidence of functional, enzymatically active endogenous HML-2 Pro being present in some tumor cell lines known to express HML-2 proteins. Our results suggest that hundreds of cellular proteins are potential substrates of HML-2 Pro. It is therefore conceivable that upregulated HML-2 transcription might lead to increased expression of active HML-2 Pro that might subsequently affect various cellular processes by degrading cellular proteins. HML-2 Pro is present in cells of some tumor types where its proteolytic activity could play a role. HML-2 Pro thus deserves further attention because cellular processes impaired by its activity potentially contribute to human diseases. In the second part of this work we investigated HML-2 IN, an enzyme that, if expressed, might locate to the cell nucleus and exhibit catalytic activities inducing DNA damage and formation of DNA double-strand breaks (DSBs). DSBs are a severe type of DNA damage that may contribute to genome instability and tumor development. We aimed at evaluating whether HML-2 IN causes DNA damage resulting in formation of DSBs. Following transient expression of HA-tagged wild-type and mutant HML-2 IN in HeLa cells, we performed immunofluorescence assays for monitoring HML-2 IN cellular localization through HA- tag detection as well as formation of DSBs through analysis of 53BP1 foci. Transiently expressed HML-2 IN localized to nuclei but we did not observe significant differences regarding 53BP1 foci when comparing cells expressing wild-type HML-2 IN and controls. Although our results point towards HML-2 IN not causing DSBs when expressed in HeLa cells the experimental approach that we established can be considered a valuable starting point for further, more comprehensive investigations.Das menschliche Genom enthält zahlreiche Sequenzen, die von ehemaligen retroviralen Infektionen stammen, sogenannte humane endogene Retroviren (HERVs). Die HERVs der HERV-K (HML-2) Gruppe, kurz HML-2, kodieren verschiedene retrovirale Proteine, die, wenn sie exprimiert werden, die Zellbiologie beeinflussen können. Die HML-2 Transkription wird in verschiedenen Krankheitskontexten hochreguliert, einschließlich der Expression HML-2-kodierter Proteinen. Einige HML-2 Proteine sind eventuell an der Entwicklung bestimmter Krankheiten beteiligt, obwohl definitive Assoziationen mit solchen Krankheiten noch nicht beschrieben sind. Diese Arbeit konzentrierte sich auf HML-2 Protease (Pro) und HML-2 Integrase (IN), zwei HML-2 kodierte Proteine, die bisher wenig Beachtung fanden, obwohl ihre enzymatischen Aktivitäten im Prinzip schädliche Auswirkungen auf die Zelle haben können. Im ersten Teil dieser Arbeit haben wir HML-2 Pro untersucht. Es ist bekannt, dass neben den retroviralen Virion-bildenden Proteinen einige retrovirale Aspartylproteasen, darunter die HIV-1- Protease, auch noch zelluläre Proteine spalten können. Es bestand die Frage, ob die proteolytische Aktivität von HML-2 Pro auch zelluläre Proteine spalten kann. Durch die Spaltung von zellulären Proteinen kann HML-2 Pro zelluläre Prozesse beeinträchtigen und somit eine Rolle bei der Krankheitsentwicklung spielen. Das Hauptziel dieses Teils der Arbeit war die Identifizierung von zellulären Proteinen, die von HML-2 Pro prozessiert wurden. Wir führten auch erste Experimente durch, die die HML-2 Pro Aktivität gegen zelluläre Proteine in einer zellulären Umgebung dokumentierten, und lieferten Einblicke in zelluläre Prozesse, die möglicherweise durch die HML-2 Pro Aktivität beeinflusst werden könnten. HML-2 Pro wurde erfolgreich gereinigt und die Reaktionsbedingungen zum Testen seiner Aktivität in vitro wurden optimiert. Gereinigtes HML-2 Pro wurde mit zellulären nativen Proteinen aus HeLa- Zellen inkubiert. Unter Verwendung der terminalen Aminisotopenmarkierung von Substraten (TAILS) identifizierten wir zelluläre Proteine, die durch HML-2 Pro prozessiert wurden. Das TAILS- Experiment und die anschließende Analyse der Rohdaten ergaben eine Liste von 872 humanen Proteinen als mutmaßliche zelluläre Substrate von HML-2 Pro. Die dentifizierten Proteine wurden unter V erwendung geeigneter bioinformatischer Werkzeuge charakterisiert. Die identifizierten Proteine konnten verschiedenen Zellkompartimenten und verschiedenen, oft krankheitsrelevanten Zellprozessen zugeordnet werden. Wir verifizierten ferner durch zusätzliche Experimente die Prozessierung ausgewählter Kandidatenproteine durch HML-2 Pro in vitro und in vivo. Eine solche Prozessierung durch HML-2 Pro wurde für 9 von 14 ausgewählten Kandidatenproteinen in vitro bestätigt. Weitere Verifikationsexperimente zeigten in vivo die Prozessierung von zellulären Proteinen durch HML-2 Pro in einer zellulären Umgebung. Die Größen der für einige der getesteten Proteine beobachteten Prozessierungsprodukte stimmten hierbei mit den von TAILS vorhergesagten Produktgrößen überein, was die TAILS-Ergebnisse bestätigte. Wir dokumentierten weiter den Zelltod und die Aktivierung apoptotischer Prozesse während der Überexpression von HML-2 Pro und lieferten damit erste experimentelle Beweise, dass zelluläre Prozesse durch die HML-2 Pro-Aktivität beeinflusst werden. Schließlich erhielten wir vorläufige Beweise dafür, dass funktionelles, enzymatisch aktives endogenes HML-2 Pro in einigen Tumorzelllinien, von denen HML-2 Proteinexpression bekannt ist, ebenso exprimiert wird. Unsere Ergebnisse legen nahe, dass Hunderte von zellulären Proteinen potenzielle Substrate von HML- 2 Pro sind. Es ist daher denkbar, dass hochregulierte HML-2 Transkription zu einer erhöhten Expression von aktivem HML-2 Pro führt, die anschließend verschiedene zelluläre Prozesse durch Abbau zellulärer Proteine beeinflussen könnte. HML-2 Pro ist in Zellen einiger Tumortypen vorhanden, in denen seine proteolytische Aktivität eine Rolle spielen könnte. HML-2 Pro verdient daher weitere Aufmerksamkeit, da zelluläre Prozesse, die durch seine Aktivität beeinträchtigt werden, möglicherweise zu menschlichen Krankheiten beitragen. Im zweiten Teil dieser Arbeit untersuchten wir HML-2 IN, ein Enzym, das bei Expression im Zellkern lokalisieren und katalytische Aktivitäten aufweisen könnte, die eine DNA-Schädigung und die Bildung von DNA-Doppelstrangbrüchen (DSBs) induzieren. DSBs sind eine schwerwiegende Art von DNA- Schäden, die zur Instabilität des Genoms und zur Tumorentwicklung beitragen können. Wir wollten untersuchen, ob HML-2 IN DNA-Schäden verursacht, die zur Bildung von DSBs führen. Nach der Expression von HA-markiertem Wildtyp- und mutiertem HML-2 IN in HeLa-Zellen führten wir Immunfluoreszenzexperimente zur Untersuchung der HML-2 IN-Zelllokalisation durch HA-Tag- Detektion sowie zur Analyse der Bildung von DSBs durch Analyse von 53BP1-Foci durch. Transient exprimiertes HML-2 IN lokalisiert im Zellkern, jedoch beobachteten wir keine signifikanten Unterschiede in Bezug auf 53BP1-Foci im Vergleich von Zellen, die Wildtyp-HML-2 IN oder Kontrollen exprimierten. Obwohl unsere Ergebnisse darauf hindeuten, dass HML-2 IN bei Expression in HeLa-Zellen keine DSBs verursacht, kann der von uns etablierte experimentelle Ansatz als wertvoller Ausgangspunkt für weitere, umfassendere Untersuchungen angesehen werden

CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cell surface and can be purified from cell conditionated media. These vesicles are known to be involved in cell migration and tumor progression. We recently demonstrated that FGF-2, a growth factor devoid of the classical signaling sequence, is secreted as a component of these vesicles. In order to analyze how molecules are clustered in shed vesicles we followed their intracellular movements by immunofluorescence techniques. The role of cytoskeletal components was analyzed using molecules such as paclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In the absence of serum, no clear localization of FGF-2 was observed. After serum addition, FGF-2 was localized partially in the nucleus and nucleolus, and partially in granules near the plasma membrane. Nocodazole and paclitaxol, which interfere with microtubular organization, inhibit FGF-2 nuclear localization but do not appear to modify FGF-2 movements toward the plasma membrane. Cytocalasine, which interferes with actin polymerization, decreases FGF-2 clustering in granules localized near the cell membrane. In summary, microtubular organization seems to be required for FGF-2 nuclear localization while actin filaments appear to be needed for FGF-2 translocation toward the plasma membrane. In a different set of experiments, we analyzed localization of neutral ceramidase (ncDase) and of Sphingosine Kinase (SphK). Ceramidase catalyzes ceramide hydrolysis giving rise to sphingosine, which in turn can be phosphorilated to S1P by SphK. Sp1P is an important signaling molecule involved in induction of cell migration and apoptosis. SphK-1 was known to be shed into the extracellular medium by an unconventional mechanism, we hypothesized that shed vesicles could vehicle its release. We therefore analyzed the localization of membrane-bound isoforms of ceramidase (ncDase) and of SphK (SphK-1 and SphK-2) by western blotting and Immunofluorescence techniques. Immunolocalization showed that ncDase is located into the plasma membrane and in cellular extensions. The concentration of ncDase was found to be higher in extracts of shed vesicles than in cell extracts. SphK-1 was found to be localized in plasma membrane and in vesicles, which appear to be enriched in this enzyme. SphK-2 was preferentially located in the nucleus and it was not detected in vesicles. In conclusion, ncDase and SphK were found to be clustered in shed vesicles. In order to analyze the role of SphK-1, either in the shedding phenomenon or in vesicle functions, we used transiently transfected SK-Hep1 cells, which overexpress SphK or express a non-functional mutant of this enzyme

Shed vesicles are involved in the release of some leaderless proteins.

Most proteins destined for secretion in the extracellular matrix are characterized by the presence of N-terminal signal peptides which direct their translocation into the endoplasmic reticulum, they are subsequently transferred to the Golgi apparatus and then secreted in the extracellular space. A growing number of secreted proteins, are being identified which, however, lack signal peptides allowing their entrance into the endoplasmic reticulum. They include the inflammatory cytokine interleukin 1b, galactins, macrophage migration inhibitory factor (MIF), acid and basic fibroblast growth factors (FGF-1, FGF-2) and Sphingosine kinase1(SphK-1). These proteins are secreted from the cell by unconventional processes which are the subject of numerous studies. Several types of normal and tumor cells can release in the extracellular medium microvesicles, called esovesicles, which result from budding of their plasma membranes. The vesicle diameter ranges between 100nm and 1000nm, the vesicle composition and function depends on the kind of the cell from which they have been produced. We already reported that FGF-2, a secreted lectin that transmits proangiogenic signals, and which is recognized as a potential oncoprotein able to modulate tumour growth and malignancy (Sorensen et al 2006), is released from SkHep1 cells, and from transfected NIH 3T3 cells through vesicle shedding (Taverna et al.2003). Now we are trying to elucidate the intracellular route followed by the growth factor from the site of synthesis to vesicles budding from the cell membrane. Actin filaments appear to be a binary for this intracellular trafficking. After 6h of treatment with cytocalasine, a drug that interferes with actin polymerization, the amount of vesicles was in fact decreased and FGF-2 clustering in granules localized near the cell surface was avoided. On the contrary no effects were observed when cells were treated with drugs which interfere with microtubule polymerization or de-polymerization. We also observed that FGF-2 granules are not included in lipid-coated vesicles. We are also analyzing the possibility that esovesicles are involved in the secretion of another leader-less signalling protein: Sphingosine kinase1 (SphK1). SphK1 has been shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Spiegel et al. 2003). SphK1 is primarily localized in the cytosol; when a signal induces the phosphorylation of Ser 225 of SphK1 through the activation of MAPK and ERK1/2, the molecule is translocated in plasma membranes and the involvement of actin filaments in its targeting has been reported (Pitson et. al. 2003). Three SphK1 isoforms having a different number of amino acids (384, 398 and 470) have been identified, we found that extracellular vesicles are enriched in the 47kDa isoform. SphK assays with TLC confirm that the enzyme is present in shed vesicles and that it has enzymatic activity. The substrate Sphingosine is also present in esovesicles therefore shed vesicles are likely to be a site of Sphingosine 1 Phosphate production

3D polylactide-based scaffolds for studying human hepatocarcinoma processes in vitro

We evaluated the combination of leaching techniques and melt blending of polymers and particles for the preparation of highly interconnected three-dimensional polymeric porous scaffolds for in vitro studies of human hepatocarcinoma processes. More specifically, sodium chloride and poly(ethylene glycol) (PEG) were used as water-soluble porogens to form porous and solvent-free poly(L,D-lactide) (PLA)-based scaffolds. Several characterization techniques, including porosimetry, image analysis and thermogravimetry, were combined to improve the reliability of measurements and mapping of the size, distribution and microarchitecture of pores. We also investigated the effect of processing, in PLA-based blends, on the simultaneous bulk/surface modifications and pore architectures in the scaffolds, and assessed the effects on human hepatocarcinoma viability and cell adhesion. The influence of PEG molecular weight on the scaffold morphology and cell viability and adhesion were also investigated. Morphological studies indicated that it was possible to obtain scaffolds with well-interconnected pores of assorted sizes. The analysis confirmed that SK-Hep1 cells adhered well to the polymeric support and emitted surface protrusions necessary to grow and differentiate three-dimensional systems. PEGs with higher molecular weight showed the best results in terms of cell adhesion and viability

3d collagen hydrogel promotes in vitro langerhans islets vascularization through ad-mvfs angiogenic activity

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 µm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation

Water-borne Polymeric Nanoparticles for Glutathione-Mediated Intracellular Delivery of Anticancer Drugs.

A new family of water-borne, biocompatible and carboxyl- functionalized nanogels was developed for glutathione- mediated delivery of anticancer drugs. Poly(N-vinyl- pyrrolidone)-co-acrylic acid nanogels were generated by e- beam irradiation of aqueous solutions of a crosslinkable polymer, using industrial-type linear accelerators and set- ups. Nanogels physico-chemical properties and colloidal stability, in a wide pH range, were investigated. In vitro cell studies proved that the nanogels are fully biocompatible and able to quantitatively bypass cellular membrane. An anticancer drug, doxorubicin (DOX), was linked to the carboxyl groups of NGs through a spacer containing a disulphide cleavable linkage. In vitro release studies showed that glutathione is able to trigger the release of DOX through the reduction of the S-S linkage at a concentration comparable to its levels in the cytosol

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

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses

Use of Modified 3D Scaffolds to Improve Cell Adhesion and Drive Desired Cell Responses.

In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement.In the most common approach of tissue engineering, a polymeric scaffold with a well-defined architecture has emerged as a promising platform for cells adhesion and guide their proliferation and differentiation into the desired tissue or organ. An ideal model for the regeneration should mimic clinical conditions of tissue injury, create a permissive microenvironment for diffusion of nutrients, gases and growth factors and permit angiogenesis. In this work, we used a 3D support made of synthetic resorbable polylactic acid (PLLA), which has considerable potential because of its well-known biocompatibility and biodegradability. One of the factors that influence cell adhesion to the scaffold is its porosity degree, but surface properties represent the main driving forces that influence the composition and orientation of proteins that will be absorbed onto material surfaces. We used scaffolds in which it was possible to control pore size and that had undergone on type-I collagen treatment, to mimic the extra cellular matrix, or plasma enhanced chemical vapor deposition (PE-CVD) combined with plasma treatment, in order to modify surface chemistry of the material. Our results show different cell affinity in non-treated scaffolds compared to type-I collagen or plasma modified ones. These surface changes are of considerable interest for tissue engineering and other areas of biomaterials science, where it can be useful to improve the surface of biomedical polymers to facilitate the colonization of the structure by the cells and obtain a more rapid regeneration or tissue replacement. Copyright © 2012, AIDIC Servizi S.r.l