Sandrine Vadon-Le Goff

Inhibition of arginase in rat and rabbit alveolar macrophages by Nω-hydroxy-D,L-indospicine, effects on L-arginine utilization by nitric oxide synthase

Alveolar macrophages (AMΦ) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate, L‐arginine. The present study tested whether two recently described potent inhibitors of liver arginase (Nω‐hydroxy‐D,L‐indospicine and 4‐hydroxyamidino‐D,L‐phenylalanine) might also inhibit arginase in AMΦ and whether inhibition of arginase might affect L‐arginine utilization by iNOS. AMΦ obtained by broncho‐alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3×106 cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [*H]‐L‐arginine uptake (37 kBq, 0.1 μM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AMΦ were incubated for 1 h with [*H]‐L‐arginine (37 kBq, 0.1 μM) and the accumulation of [*H]‐L‐citrulline (NOS activity) and [*H]‐L‐ornithine (arginase activity) was determined. During 1 h incubation of rabbit AMΦ with [*H]‐L‐arginine, no [*H]‐L‐citrulline, but significant amounts of [*H]‐L‐ornithine (150 d.p.m.×1000) were formed. Nω‐hydroxy‐D,L‐indospicine and 4‐hydroxyamidino‐D,L‐phenylalanine, present during incubation, concentration‐dependently reduced [*H]‐L‐ornithine formation (IC50: 2 and 45 μM, respectively). Nω‐hydroxy‐D,L‐indospicine (up to 100 μM) had no effect on [*H]‐L‐arginine uptake into rabbit AMΦ, whereas 4‐hydroxyamidino‐D,L‐phenylalanine caused a concentration‐dependent inhibition (IC50: 300 μM). Rat AMΦ, cultured in the absence of LPS, formed significant amounts of [*H]‐L‐citrulline and [*H]‐L‐ornithine (133 and 212 d.p.m.×1000, respectively) when incubated for 1 h with [*H]‐L‐arginine. When AMΦ had been cultured in the presence of 0.1 or 1 μg ml−1 LPS, the formation of [*H]‐L‐citrulline was enhanced by 37±8.3 and 99±12% and that of [*H]‐L‐ornithine reduced by 21±8.7 and 70±2.5%, respectively. In rat AMΦ, cultured in the absence or presence of LPS, Nω‐hydroxy‐D,L‐indospicine (10 and 30 μM) greatly reduced formation of [*H]‐L‐ornithine (by 80–95%) and this was accompanied by increased formation of [*H]‐L‐citrulline. However, only 20–30% of the [*H]‐L‐arginine not metabolized to [*H]‐L‐ornithine after inhibition of arginase was metabolized to [*H]‐L‐citrulline, when the AMΦ had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AMΦ had been cultured in the presence of LPS (i.e. high level of iNOS), all the [*H]‐L‐arginine not metabolized by the inhibited arginase was metabolized to [*H]‐L‐citrulline. In conclusion, Nω‐hydroxy‐D,L‐indospicine is a potent and specific inhibitor of arginase in AMΦ. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L‐arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.

Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity

Cholinergic airway constriction is functionally antagonized by agonist‐induced constitutive nitric oxide synthase (cNOS)‐derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L‐arginine to L‐ornithine and urea, use L‐arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea‐pig tracheal tube preparation, we investigated the modulation of methacholine‐induced airway constriction by the recently developed, potent and specific arginase inhibitor NΩ‐hydroxy‐nor‐L‐arginine (nor‐NOHA). Intraluminal (IL) administration of nor‐NOHA caused a concentration‐dependent inhibition of the maximal effect (Emax) in response to IL methacholine, which was maximal in the presence of 5 μM nor‐NOHA (Emax=31.2±1.6% of extraluminal (EL) 40 mM KCl‐induced constriction versus 51.6±2.1% in controls, P<0.001). In addition, the pEC50 (−log10 EC50) was slightly but significantly reduced in the presence of 5 μM nor‐NOHA. The inhibition of Emax by 5 μM nor‐NOHA was concentration‐dependently reversed by the NOS inhibitor NΩ‐nitro‐L‐arginine methyl ester (L‐NAME), reaching an Emax of 89.4±7.7% in the presence of 0.5 mM L‐NAME (P<0.01). A similar Emax in the presence of 0.5 mM L‐NAME was obtained in control preparations (85.2±9.7%, n.s.). In the presence of excess of exogenously applied L‐arginine (5 mM), 5 μM nor‐NOHA was ineffective (Emax=33.1±5.8 versus 31.1±7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine‐induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L‐arginine. This finding may represent an important novel regulation mechanism of airway reactivity.

Processing of Procollagen III by Meprins: New Players in Extracellular Matrix Assembly?

Meprins α and β, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins α and β release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin α is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin α increases and meprin β begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Metalloproteases meprin α and meprin β are C- and N-procollagen proteinases important for collagen assembly and tensile strength

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a−/− and Mep1b−/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

Procollagen C-proteinase Enhancer Stimulates Procollagen Processing by Binding to the C-propeptide Region Only

Background: Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular glycoprotein that increases activity of certain zinc metalloproteinases involved in tissue development and repair. Results: PCPE-1 binds uniquely to the C-propeptide region of the procollagen molecule. Conclusion: PCPE-1 enhances proteolysis by binding solely to the procollagen C-propeptides. Significance: These data may lead to future applications in the development of antifibrotic therapies. Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

Procollagen C-proteinase enhancer grasps the stalk of the C-propeptide trimer to boost collagen precursor maturation

Tight regulation of collagen fibril deposition in the extracellular matrix is essential for normal tissue homeostasis and repair, defects in which are associated with several degenerative or fibrotic disorders. A key regulatory step in collagen fibril assembly is the C-terminal proteolytic processing of soluble procollagen precursors. This step, carried out mainly by bone morphogenetic protein-1/tolloid-like proteinases, is itself subject to regulation by procollagen C-proteinase enhancer proteins (PCPEs) which can dramatically increase bone morphogenetic protein-1/tolloid-like proteinase activity, in a substrate-specific manner. Although it is known that this enhancing activity requires binding of PCPE to the procollagen C-propeptide trimer, identification of the precise binding site has so far remained elusive. Here, use of small-angle X-ray scattering provides structural data on this protein complex indicating that PCPE binds to the stalk region of the procollagen C-propeptide trimer, where the three polypeptide chains associate together, at the junction with the base region. This is supported by site-directed mutagenesis, which identifies two highly conserved, surface-exposed lysine residues in this region of the trimer that are essential for binding, thus revealing structural parallels with the interactions of Complement C1r/C1s, Uegf, BMP-1 (CUB) domain-containing proteins in diverse biological systems such as complement activation, receptor signaling, and transport. Together with detailed kinetics and interaction analysis, these results provide insights into the mechanism of action of PCPEs and suggest clear strategies for the development of novel antifibrotic therapies.

Strong cooperativity and loose geometry between CUB domains are the basis for procollagen C-proteinase enhancer activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

Sizzled Is Unique among Secreted Frizzled-related Proteins for Its Ability to Specifically Inhibit Bone Morphogenetic Protein-1 (BMP-1)/Tolloid-like Proteinases

Background: Xenopus and zebrafish BMP-1/tolloid-like proteinases (BTPs) are inhibited by sizzled, a secreted frizzled-related protein (sFRP) not present in mammals. Results: Xenopus sizzled is a very potent inhibitor of human BTPs, whereas mammalian sFRPs have no effect. Conclusion: Regulation of BTP activity by sFRPs is not conserved in mammals. Significance: Sizzled is the most potent exogenous inhibitor of human BTPs. BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a Ki of 1.5 ± 0.5 nm, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-β signaling as primary targets

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N‐proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N‐terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF‐β binding protein 1, TGF‐β RIII, and dickkopf‐related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF‐β activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for non‐polar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.—Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon‐Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella‐Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF‐β signaling as primary targets. FASEB J. 30, 1741–1756 (2016). www.fasebj.org

Proteolytic control of TGF-β co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics.

The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-β superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-β co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-β was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-β co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi:10.6019/PXD000786.