Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used surface-spanning enrichment with click-sugars (SUSPECS) proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding

Quantitative Proteomics Reveals That ADAM15 Can Have Proteolytic-Independent Functions in the Steady State

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies

Effects of oral administration of silymarin in a juvenile murine model of non-alcoholic steatohepatitis

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been de\ufb01ned so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid pro\ufb01le, transaminases, HOMA-IR, steatosis, in\ufb02ammation, \ufb01brosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic lifestyle changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a signi\ufb01cant improvement in glycemia, visceral fat, lipid pro\ufb01le, and liver \ufb01brosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic in\ufb02ammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the bene\ufb01cial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH

Effects of oral administration of silymarin in a juvenile murine model of non-alcoholic steatohepatitis

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) in adolescents is challenging the global care system. No therapeutic strategies have been defined so far, and changes in the lifestyle remain the only alternative. In this study, we assessed the protective effects of silymarin in a juvenile non-alcoholic steatohepatitis (NASH) model and the in vitro effects on fat-laden human hepatocytes. C57Bl/6 mice were exposed to HFHC diet immediately after weaning. After eight weeks, animals showed histological signs of NASH. Silymarin was added to the HFHC diet, the treatment continued for additional 12 weeks and the effects on BMI, hepatomegaly, visceral fat, lipid profile, transaminases, HOMA-IR, steatosis, inflammation, fibrosis, oxidative stress, and apoptosis were determined. The switch from HFHC to control diet was used to mimic lifestyle changes. In vitro experiments were performed in parallel in human hepatocytes. HFHC diet supplemented with silymarin showed a significant improvement in glycemia, visceral fat, lipid profile, and liver fibrosis. Moreover, it reduced (both in vitro and in vivo) ALT, hepatic inflammation, oxidative stress, and apoptosis. Lifestyle changes restored the control group parameters. The data presented show the beneficial effects of the oral administration of silymarin in the absence of changes in the dietary habits in a juvenile model of NASH

Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used “surface-spanning enrichment with click-sugars (SUSPECS)” proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding

La conducibilit\ue0 elettrica come strumento per stimare lo sviluppo del reticolo ipogeo

Una delle principali sfide nella ricerca speleologica \ue8 quella di stimare in modo il pi\uf9 possibile accurato l\u2019entit\ue0 (dimensione, estensione e sviluppo) e la localizzazione del reticolo ipogeo presente in un\u2019area carsica. Quando l\u2019attivit\ue0 esplorativa classica non risolve tutte le problematiche si utilizzano tecniche indirette quali per esempio la geofisica o le analisi idrogeologiche. In quest\u2019ottica nel 2015 \ue8 partita una proficua collaborazione tra gli speleologi del Gruppo Speleologico Monfalconese \u201cAmici del Fante\u201d, speleologi del Gruppo Speleologico \u201cTalpe del Carso\u201d - Jamarski Klub \u201cKraski Krti\u201d, studiosi indipendenti e ricercatori del dipartimento di Matematica e Geoscienze volta alla comprensione dei deflussi sotterranei e delle modalit\ue0 di ricarica/esaurimento del settore occidentale del Carso Classico. In questi ultimi 3 anni \ue8 stata sviluppata una metodologia che attraverso l\u2019analisi della conducibilit\ue0 elettrica delle acque superficiali e sotterranee presenti nell\u2019area di studio permette di individuare l\u2019origine e la provenienza delle diverse tipologie di acque presenti. La scelta del parametro conducibilit\ue0 elettrica non deriva solo dalla sua significativit\ue0, ma anche dal fatto che essa rappresenta un parametro di facile acquisizione e che non necessita di complesse e costose metodologie di analisi. Dato che la ricarica dell\u2019acquifero \ue8 dovuta principalmente a tre componenti quali le perdite dell\u2019Isonzo, l\u2019infiltrazione efficace e il contributo del sistema Reka-Timavo, da giugno 2015 ai primi mesi del 2018 sono state effettuate quasi 100 uscite sul terreno che hanno permesso di acquisire dati di conducibilit\ue0 elettrica e temperatura presso l\u2019Isonzo, il Reka, i laghi di Doberd\uf2 e Pietrarossa, le sorgenti di Sablici di Moschenizze, di Sardos e del Timavo. Nel contempo sono state installate due sonde multiparametriche sul fondo dell\u2019Abisso Samar di Riky (cat. FVG n.4709) e della Grotta del Proteo (cat. FVG n.1191) presso Sagrado (GO), che ogni 30 minuti acquisiscono informazioni sulla conducibilit\ue0 elettrica, la temperatura e il livello delle acque. L\u2019analisi dei dati ha permesso di individuare i valori caratteristici delle tre componenti principali di ricarica dell\u2019acquifero: l\u2019Isonzo con un valore di 240 uS/cm, le acque carsiche dovute al contributo delle precipitazioni con 470 uS/cm e il Reka con 310 uS/cm. Utilizzando questi valori \ue8 stato possibile quantificare il contributo delle diverse componenti sia in corrispondenza delle sorgenti che dei laghi carsici nei diversi regimi idrogeologici. Nel contempo l\u2019analisi dei dati in continuo ha permesso di stimare le modalit\ue0 di ricarica ed esaurimento del sistema ipogeo. Il lavoro che si presenta \ue8 stato realizzato grazie anche al contributo della Federazione Speleologica Isontina, dell\u2019Universit\ue0 degli Studi di Trieste e della Fondazione Carigo di Gorizia

An In Vitro Model of Glioma Development

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion

Quantitative proteomics reveals that ADAM15 can have proteolytic-independent functions in the steady state

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells