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

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

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

LRP-1-dependent and -independent endocytic pathways of TIMP-3

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an endogeneous inhibitor of metalloproteinases (MMPs), adamalysins (ADAMs) and adamalysins with thrombospondin motifs (ADAMTSs), and consequently TIMP-3 is an important regulator of extracellular matrix (ECM) turnover. It has been shown that TIMP-3 levels in the tissue are regulated post-translationally by endocytosis. This study aimed to characterize the mechanism of TIMP-3 endocytosis in more detail. In this thesis, I demonstrated that TIMP-3 is endocytosed and degraded by a number of cell types including chondrocytes, fibroblasts, and monocytes. I found that the endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1) plays a major role in TIMP-3 endocytosis. Nevertheless, I found that TIMP-3 can also be internalized by LRP-1-null cells, indicating that an LRP-1-indepentent endocytic pathway also occurs. CHO-cells lacking heparin sulphate proteoglycans (HSPGs) efficiently endocytosed TIMP-3, indicating that HSPGs are not involved in its endocytosis. Shedding of LRP-1 has been shown to reduce cell surface levels of the receptor, and hence to modulate endocytosis of its ligands. I demonstrated that LRP-1 shedding leads to accumulation of soluble LRP-1 (sLRP-1) in the medium of chondrosarcoma cells. sLRP-1 interacted with TIMP-3 and inhibited its endocytosis, but had no effect on TIMP-3 inhibition of target metalloproteinases, indicating that sLRP-1 negatively regulates TIMP-3 endocytosis. TIMP-3 endocytosis was not affected by addition of GM6001, which inhibits interaction with target metalloproteinases, indicating that TIMP-3 can be internalized independently. Internalization of TIMP-3 in complex with a number of metalloproteinases occurred with the same kinetics as that of TIMP-3 alone. Furthermore, TIMP-3 promoted the endocytosis of MMP-1, suggesting that TIMP-3 can mediate the scavenging of metalloproteinases by cells. The study thus identifies LRP-1 as a central mediator of TIMP-3 endocytosis, and indicates that this process regulates levels of TIMP-3 in the ECM, thus contributing to the maintenance of tissue homeostasis

Tissue Inhibitor of Metalloproteases 3 (TIMP-3): In Vivo Analysis Underpins Its Role as a Master Regulator of Ectodomain Shedding

The proteolytical cleavage of transmembrane proteins with subsequent release of their extracellular domain, so-called ectodomain shedding, is a post-translational modification that plays an essential role in several biological processes, such as cell communication, adhesion and migration. Metalloproteases are major proteases in ectodomain shedding, especially the disintegrin metalloproteases (ADAMs) and the membrane-type matrix metalloproteases (MT-MMPs), which are considered to be canonical sheddases for their membrane-anchored topology and for the large number of proteins that they can release. The unique ability of TIMP-3 to inhibit different families of metalloproteases, including the canonical sheddases (ADAMs and MT-MMPs), renders it a master regulator of ectodomain shedding. This review provides an overview of the different functions of TIMP-3 in health and disease, with a major focus on the functional consequences in vivo related to its ability to control ectodomain shedding. Furthermore, herein we describe a collection of mass spectrometry-based approaches that have been used in recent years to identify new functions of sheddases and TIMP-3. These methods may be used in the future to elucidate the pathological mechanisms triggered by the Sorsby’s fundus dystrophy variants of TIMP-3 or to identify proteins released by less well characterized TIMP-3 target sheddases whose substrate repertoire is still limited, thus providing novel insights into the physiological and pathological functions of the inhibitor

Strategies to Target ADAM17 in Disease: From Its Discovery to the iRhom Revolution

For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells

Strategies to Target ADAM17 in Disease: From Its Discovery to the iRhom Revolution

For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells

The Repertoire of Tissue Inhibitors of Metalloproteases: Evolution, Regulation of Extracellular Matrix Proteolysis, Engineering and Therapeutic Challenges

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1–4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the homologues from cnidarians, which are coeval with the origins of ECM components; protostomes (molluscs, arthropods and nematodes); and deuterostomes (echinoderms and vertebrates). Several questions, including the maintenance of the structure despite low sequence similarity and the strategies for TIMP engineering, shed light on the possibility to use recombinant TIMPs integrating unique features and binding selectivity for therapeutic applications in the treatment of inflammatory pathologies