‘A disintegrin and metalloproteases’ (ADAMs) are a family of transmembrane proteins with diverse functions in multicellular organisms. About half of the ADAMs are active metalloproteases and cleave ...numerous cell surface proteins, including growth factors, receptors, cytokines and cell adhesion proteins. The other ADAMs have no catalytic activity and function as adhesion proteins or receptors. Some ADAMs are ubiquitously expressed, others are expressed tissue specifically. This review highlights functions of ADAMs in the mammalian nervous system, including their links to diseases. The non-proteolytic ADAM11, ADAM22 and ADAM23 have key functions in neural development, myelination and synaptic transmission and are linked to epilepsy. Among the proteolytic ADAMs, ADAM10 is the best characterized one due to its substrates Notch and amyloid precursor protein, where cleavage is required for nervous system development or linked to Alzheimer’s disease (AD), respectively. Recent work demonstrates that ADAM10 has additional substrates and functions in the nervous system and its substrate selectivity may be regulated by tetraspanins. New roles for other proteolytic ADAMs in the nervous system are also emerging. For example, ADAM8 and ADAM17 are involved in neuroinflammation. ADAM17 additionally regulates neurite outgrowth and myelination and its activity is controlled by iRhoms. ADAM19 and ADAM21 function in regenerative processes upon neuronal injury. Several ADAMs, including ADAM9, ADAM10, ADAM15 and ADAM30, are potential drug targets for AD. Taken together, this review summarizes recent progress concerning substrates and functions of ADAMs in the nervous system and their use as drug targets for neurological and psychiatric diseases.
Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The ...low-density lipoprotein receptor-related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 single nucleotide polymorphisms are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 knockout mice revealed a critical, non-redundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these “hidden” specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.
Matrix metalloproteinases (MMPs) and the related families of disintegrin metalloproteinases (ADAMs) and ADAMs with thrombospondin repeats (ADAMTSs) play a crucial role in extracellular matrix (ECM) ...turnover and shedding of cell-surface molecules. The proteolytic activity of metalloproteinases is post-translationally regulated by their endogenous inhibitors, known as tissue inhibitors of metalloproteinases (TIMPs). Several MMPs, ADAMTSs and TIMPs have been reported to be endocytosed by the low-density lipoprotein receptor-related protein-1 (LRP-1). Different binding affinities of these proteins for the endocytic receptor correlate with different turnover rates which, together with differences in their mRNA expression, determines their nett extracellular levels. In this study, we used surface plasmon resonance to evaluate the affinity between LRP-1 and a number of MMPs, ADAMs, ADAMTSs, TIMPs and metalloproteinase/TIMP complexes. This identified MMP-1 as a new LRP-1 ligand. Among the proteins analyzed, TIMP-3 bound to LRP-1 with highest affinity (K
= 1.68 nM). Additionally, we found that TIMP-3 can facilitate the clearance of its target metalloproteinases by bridging their binding to LRP-1. For example, the free form of MMP-1 was found to have a K
of 34.6 nM for LRP-1, while the MMP-1/TIMP-3 complex had a sevenfold higher affinity (K
= 4.96 nM) for the receptor. TIMP-3 similarly bridged binding of MMP-13 and MMP-14 to LRP-1. TIMP-1 and TIMP-2 were also found to increase the affinity of target metalloproteinases for LRP-1, albeit to a lesser extent. This suggests that LRP-1 scavenging of TIMP/metalloproteinase complexes may be a general mechanism by which inhibited metalloproteinases are removed from the extracellular environment.
Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and ...control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the "a disintegrin and metalloprotease" (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer's disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even
from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.
Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 ...are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.
Cell surface proteolysis controls numerous biological processes including cell-cell attachment and the communication between cells. The membrane-tethered families of matrix metalloproteinases ...(MT-MMPs) and disintegrin metalloproteinases (ADAMs) are major enzymes involved in the cleavage of molecules at the cell surface, and their activity is finely regulated by their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). The biological function of a metalloproteinase closely depends on the subset of substrates that it cleaves. Similarly, molecular processes that are regulated by a specific TIMP strictly depend on its unique inhibitory profile.Herein, we describe a mass spectrometry-based method for the quantitative analysis of protein abundance in conditioned media of cultured cells that is particularly suited for substrate identification of membrane-tethered metalloproteinases and for the identification of membrane proteins whose cleavage is regulated by TIMPs. This unbiased proteomic method represents a valuable tool to investigate biological functions of metalloproteinases and TIMPs at the "omic" level.
Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this ...context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8
+
T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.
Tissue inhibitor of metalloproteinases-3 (TIMP-3) plays a key role in regulating extracellular matrix turnover by inhibiting matrix metalloproteinases (MMPs), adamalysins (ADAMs), and adamalysins ...with thrombospondin motifs (ADAMTSs). We demonstrate that levels of this physiologically important inhibitor can be regulated post-translationally by endocytosis. TIMP-3 was endocytosed and degraded by a number of cell types including chondrocytes, fibroblasts, and monocytes, and we found that the endocytic receptor low density lipoprotein receptor-related protein-1 (LRP-1) plays a major role in TIMP-3 internalization. However, the cellular uptake of TIMP-3 significantly slowed down after 10 h due to shedding of LRP-1 from the cell surface and formation of soluble LRP-1 (sLRP-1)-TIMP-3 complexes. Addition of TIMP-3 to HTB94 human chondrosarcoma cells increased the release of sLRP-1 fragments of 500, 215, 160, and 110 kDa into the medium in a concentration-dependent manner, and all of these fragments were able to bind to TIMP-3. TIMP-3 bound to sLRP-1, which was resistant to endocytosis, retained its inhibitory activity against metalloproteinases. Extracellular levels of sLRP-1 can thus increase the half-life of TIMP-3 in the extracellular space, controlling the bioavailability of TIMP-3 to inhibit metalloproteinases.
Background: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is endocytosed, but its regulatory mechanism is not well understood.
Results: TIMP-3 endocytosis occurs mainly through low density lipoprotein receptor-related protein-1 (LRP-1), but shed sLRP-1 binds TIMP-3.
Conclusion: TIMP-3-sLRP-1 complexes are retained extracellularly with metalloproteinase inhibitory activity.
Significance: LRP-1 is the master regulator of extracellular levels of TIMP-3.
Aggrecan is a major matrix component of articular cartilage, and its degradation is a crucial event in the development of osteoarthritis (OA). Adamalysin‐like metalloproteinase with thrombospondin ...motifs 5 (ADAMTS‐5) is a major aggrecan‐degrading enzyme in cartilage, but there is no clear correlation between ADAMTS‐5 mRNA levels and OA progression. Here, we report that post‐translational endocytosis of ADAMTS‐5 by chondrocytes regulates its extracellular activity. We found 2‐ to 3‐fold reduced aggrecanase activity when ADAMTS‐5 was incubated with live porcine cartilage, resulting from its rapid endocytic clearance. Studies using receptor‐associated protein (RAP), a ligand‐binding antagonist for the low‐density lipoprotein receptor‐related proteins (LRPs), and siRNA‐mediated gene silencing revealed that the receptor responsible for ADAMTS‐5 clearance is LRP‐1. Domain‐deletion mutagenesis of ADAMTS‐5 identified that the noncatalytic first thrombospondin and spacer domains mediate its endocytosis. The addition of RAP to porcine cartilage explants in culture increased the basal level of aggrecan degradation, as well as ADAMTS‐5‐induced aggrecan degradation. Notably, LRP‐1‐mediated endocytosis of ADAMTS‐5 is impaired in chondrocytes of OA cartilage, with ~90% reduction in protein levels of LRP‐1 without changes in its mRNA levels. Thus, LRP‐1 dictates physiological and pathological catabolism of aggrecan in cartilage as a key modulator of the extracellular activity of ADAMTS‐5.—Yamamoto, K., Troeberg, L., Scilabra, S. D., Pelosi, M., Murphy, C. L., Strickland, D. K., Nagase, H. LRP‐1‐mediated endocytosis regulates extracellular activity of ADAMTS‐5 in articular cartilage. FASEB J. 27, 511–521 (2013). www.fasebj.org
Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a key regulator of extracellular matrix turnover for its ability to inhibit matrix metalloproteinases (MMPs), adamalysin-like metalloproteinases ...(ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs). TIMP-3 is a secreted protein whose extracellular levels are regulated by endocytosis via the low-density-lipoprotein receptor-related protein-1 (LRP-1). In this study we developed a molecule able to “trap” TIMP-3 extracellularly, thereby increasing its tissue bioavailability. LRP-1 contains four ligand-binding clusters. In order to investigate the TIMP-3 binding site on LRP-1, we generated soluble minireceptors (sLRPs) containing the four distinct binding clusters or part of each cluster. We used an array of biochemical methods to investigate the binding of TIMP-3 to different sLRPs. We found that TIMP-3 binds to the ligand-binding cluster II of the receptor with the highest affinity and a soluble minireceptor containing the N-terminal half of cluster II specifically blocked TIMP-3 internalization, without affecting the turnover of metalloproteinases. Mass spectrometry-based secretome analysis showed that this minireceptor, named T3TRAP, selectively increased TIMP-3 levels in the extracellular space and inhibited constitutive shedding of a number of cell surface proteins. In conclusion, T3TRAP represents a biological tool that can be used to modulate TIMP-3 levels in the tissue and could be potentially developed as a therapy for diseases characterized by a deficit of TIMP-3, including arthritis.
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