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International audience; Calcium accumulation in atherosclerotic plaques predicts cardiovascular mortality, but the mechanisms responsible for plaque calcification and how calcification impacts plaque stability remain debated. Tissue-nonspecific alkaline phosphatase (TNAP) recently emerged as a promising therapeutic target to block cardiovascular calcification. In this study, we sought to investigate the effect of the recently developed TNAP inhibitor SBI-425 on atherosclerosis plaque calcification and progression. TNAP levels were investigated in ApoE-deficient mice fed a high-fat diet from 10 weeks of age and in plaques from the human ECLAGEN biocollection (101 calcified and 14 non-calcified carotid plaques). TNAP was inhibited in mice using SBI-425 administered from 10 to 25 weeks of age, and in human vascular smooth muscle cells (VSMCs) with MLS-0038949. Plaque calcification was imaged in vivo with 18F-NaF-PET/CT, ex vivo with osteosense, and in vitro with alizarin red. Bone architecture was determined with µCT. TNAP activation preceded and predicted calcification in human and mouse plaques, and TNAP inhibition prevented calcification in human VSMCs and in ApoE-deficient mice. More unexpectedly, TNAP inhibition reduced the blood levels of cholesterol and triglycerides, and protected mice from atherosclerosis, without impacting the skeletal architecture. Metabolomics analysis of liver extracts identified phosphocholine as a substrate of liver TNAP, who's decreased dephosphorylation upon TNAP inhibition likely reduced the release of cholesterol and triglycerides into the blood. Systemic inhibition of TNAP protects from atherosclerosis, by ameliorating dyslipidemia, and preventing plaque calcification.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

Although prebiotic condensations of glycerol, phosphate and fatty acids produce phospholipid esters with a racemic backbone, most experimental studies on vesicles intended as protocell models have been carried out by employing commercial enantiopure phospholipids. Current experimental research on realistic protocell models urgently requires racemic phospholipids and efficient synthetic routes for their production. Here we propose three synthetic pathways starting from glycerol or from racemic solketal (&alpha, &beta, isopropylidene-dl-glycerol) for the gram-scale production (up to 4 g) of racemic phospholipid ester precursors. We describe and compare these synthetic pathways with literature data. Racemic phosphatidylcholines and phosphatidylethanolamines were obtained in good yields and high purity from 1,2-diacylglycerols. Racemic POPC (rac-POPC, (R,S)-1-palmitoyl-2-oleoyl-3-phosphocholine), was used as a model compound for the preparation of giant vesicles (GVs). Confocal laser scanning fluorescence microscopy was used to compare GVs prepared from enantiopure (R)-POPC), racemic POPC (rac-POPC) and a scalemic mixture (scal-POPC) of (R)-POPC enriched with rac-POPC. Vesicle morphology and size distribution were similar among the different (R)-POPC, rac-POPC and scal-POPC, while calcein entrapments in (R)-POPC and in scal-POPC were significantly distinct by about 10%.

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.

Prostate cancer (PCa) is the most frequent cancer in men aged 65 and over. PCa mainly metastasizes in the bone, forming osteosclerotic lesions, inducing pain, fractures, and nerve compression. Cancer cell-derived exosomes participate in the metastatic spread, ranging from oncogenic reprogramming to the formation of pre-metastatic niches. Moreover, exosomes were recently involved in the dialog between PCa cells and the bone metastasis microenvironment. Phospholipase D (PLD) isoforms PLD1/2 catalyze the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), regulating tumor progression and metastasis. PLD is suspected to play a role in exosomes biogenesis. We aimed to determine whether PCa-derived exosomes, through PLD, interact with the bone microenvironment, especially osteoblasts, during the metastatic process. Here we demonstrate for the first time that PLD2 is present in exosomes of C4-2B and PC-3 cells. C4-2B-derived exosomes activate proliferation and differentiation of osteoblasts models, by stimulating ERK 1/2 phosphorylation, by increasing the tissue-nonspecific alkaline phosphatase activity and the expression of osteogenic differentiation markers. Contrariwise, when C4-2B exosomes are generated in the presence of halopemide, a PLD pan-inhibitor, they lose their ability to stimulate osteoblasts. Furthermore, the number of released exosomes diminishes significantly (-40%). When the PLD product PA is combined with halopemide, exosome secretion is fully restored. Taken together, our results indicate that PLD2 stimulates exosome secretion in PCa cell models as well as their ability to increase osteoblast activity. Thus, PLD2 could be considered as a potent player in the establishment of PCa bone metastasis acting through tumor cell derived-exosomes.

International audience; Tissue-nonspecific alkaline phosphatase (TNAP) is necessary for skeletal mineralization by its ability to hydrolyze the mineralization inhibitor inorganic pyrophosphate (PPi), which is mainly generated from extracellular ATP by ectonucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Since children with TNAP deficiency develop bone metaphyseal auto-inflammations in addition to rickets, we hypothesized that TNAP also exerts anti-inflammatory effects relying on the hydrolysis of pro-inflammatory adenosine nucleotides into the anti-inflammatory adenosine. We explored this hypothesis in bone metaphyses of 7-day-old Alpl+/− mice (encoding TNAP), in mineralizing hypertrophic chondrocytes and osteoblasts, and non-mineralizing mesenchymal stem cells (MSCs) and neutrophils, which express TNAP and are present, or can be recruited in the metaphysis. Bone metaphyses of 7-day-old Alpl+/− mice had significantly increased levels of Il-1β and Il-6 and decreased levels of the anti-inflammatory Il-10 cytokine as compared with Alpl+/+ mice. In bone metaphyses, murine hypertrophic chondrocytes and osteoblasts, Alpl mRNA levels were much higher than those of the adenosine nucleotidases Npp1, Cd39 and Cd73. In hypertrophic chondrocytes, inhibition of TNAP with 25 μM of MLS-0038949 decreased the hydrolysis of AMP and ATP. However, TNAP inhibition did not significantly modulate ATP- and adenosine-associated effects in these cells. We observed that part of TNAP proteins in hypertrophic chondrocytes was sent from the cell membrane to matrix vesicles, which may explain why TNAP participated in the hydrolysis of ATP but did not significantly modulate its autocrine pro-inflammatory effects. In MSCs, TNAP did not participate in ATP hydrolysis nor in secretion of inflammatory mediators. In contrast, in neutrophils, TNAP inhibition with MLS-0038949 significantly exacerbated ATP-associated activation and secretion of IL-1β, and extended cell survival. Collectively, these results demonstrate that TNAP is a nucleotidase in both hypertrophic chondrocytes and neutrophils, and that this nucleotidase function is associated with autocrine effects on inflammation only in neutrophils.

It is an open question how the chemical structure of prebiotic vesicle-forming amphiphiles complexified to produce robust primitive compartments that could safely host foreign molecules. Previous work suggests that comparingly labile vesicles composed of plausibly prebiotic fatty acids were eventually chemically transformed with glycerol and a suitable phosphate source into phospholipids that would form robust vesicles. Here we show that phosphatidic acid (PA) and phosphatidylethanolamine (PE) lipids can be obtained from racemic dioleoyl glycerol under plausibly prebiotic phosphorylation conditions. Upon in situ hydration of the crude phosphorylation mixtures only those that contained rac-DOPA (not rac-DOPE) generated stable giant vesicles that were capable of encapsulating water-soluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction side-products (identified by IR and MS and quantified by 1H NMR) acted as co-surfactants and facilitated vesicle formation. To mimic the compositional variation of such primitive lipid mixtures, self-assembly of a combinatorial set of the above amphiphiles was tested, revealing that too high dioleoyl glycerol contents inhibited vesicle formation. We conclude that a decisive driving force for the gradual transition from unstable fatty acid vesicles to robust diacylglyceryl phosphate vesicles was to avoid the accumulation of unphosphorylated diacylglycerols in primitive vesicle membranes.

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Osteoblasts initiate bone mineralization by releasing matrix vesicles (MVs) into the extracellular matrix (ECM). MVs promote the nucleation process of apatite formation from Ca2+ and Pi in their lumen and bud from the microvilli of osteoblasts during bone development. Tissue non-specific alkaline phosphatase (TNAP) as well as annexins (among them, AnxA6) are abundant proteins in MVs that are engaged in mineralization. In addition, sarcoma proto-oncogene tyrosine-protein (Src) kinase and Rho-associated coiled-coil (ROCK) kinases, which are involved in vesicular transport, may also regulate the mineralization process. Upon stimulation in osteogenic medium containing 50 &mu, g/mL of ascorbic acid (AA) and 7.5 mM of &beta, glycerophosphate (&beta, GP), human osteosarcoma Saos-2 cells initiated mineralization, as evidenced by Alizarin Red-S (AR-S) staining, TNAP activity, and the partial translocation of AnxA6 from cytoplasm to the plasma membrane. The addition of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PP2), which is an inhibitor of Src kinase, significantly inhibited the mineralization process when evaluated by the above criteria. In contrast, the addition of (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide hydrochloride (Y-27632), which is an inhibitor of ROCK kinase, did not affect significantly the mineralization induced in stimulated Saos-2 cells as denoted by AR-S and TNAP activity. In conclusion, mineralization by human osteosarcoma Saos-2 cells seems to be differently regulated by Src and ROCK kinases.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca(2+)) and inorganic phosphate (P(i)) within their lumen and forming a nucleation core (NC). After further sequestration of Ca(2+) and P(i), the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca(2+)) and non-mineralizing (–Ca(2+)) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca(2+). This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.

BACKGROUND: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100–300 nm diameter harboring the biochemical machinery needed to induce mineralization. SCOPE OF THE REVIEW: The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MAJOR CONCLUSIONS: MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. GRAPHICAL ABSTRACT LEGEND: Matrix vesicles are extracellular vesicles that bind to collagen and can induce formation of apatitic mineral during physiological and ectopic mineralization. Lipid and protein compositions in matrix vesicles resemble those of lipid rafts. Mechanisms of the biogenesis of matrix vesicles and processes leading to mineral/apatite formation are still unclear. Proteoliposomes can serve as biomimetic models to understand matrix vesicle-mediated mineralization.

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM). Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions. The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

International audience; Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1) all of the studied polyphenols interact with albumin; (2) while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM) with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM) with also a stoichiometry of one per monomer; (3) at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4) resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body.

International audience; The involvement of membrane-bound peptides and the influence of protein conformations in several neurodegenerative diseases lead us to analyze the interactions of model peptides with artificial membranes. Two model peptides were selected. The first one, an alanine-rich peptide, K3A18K3, was shown to be in alpha-helix structures in TFE, a membrane environment-mimicking solvent, while it was mostly beta-sheeted in aqueous buffer as revealed by infrared spectroscopy. The other, alamethicin, a natural peptide, was in a stable alpha-helix structure. To determine the role of the peptide conformation on the nature of its interactions with lipids, we compared the structure and topology of the conformational-labile peptide K3A18K3 and of the alpha-helix rigid alamethicin in both aqueous and phospholipid environments (Langmuir monolayers and multilamellar vesicles). K3A18K3 at the air-water interface showed a pressure-dependent orientation of its beta-sheets, while the alpha-helix axis of alamethicin was always parallel to the interface, as probed by polarization modulation infrared reflection absorption spectroscopy. The beta-sheeted K3A18K3 peptide was uniformly distributed into DPPC condensed domains, while the helical-alamethicin insertion distorted the DPPC condensed domains, as evidenced by Brewster angle microscopy imaging of the air/interface. The beta-sheeted K3A18K3 interacted with DMPC multilamellar vesicles via hydrophilic interactions with polar heads and the helical-alamethicin via hydrophobic interactions with alkyl chains, as shown by infrared spectroscopy and solid state NMR. Our findings are consistent with the prevailing assumption that the conformation of the peptide predetermines the mode of interaction with lipids. More precisely, helical peptides tend to be inserted via hydrophobic interactions within the hydrophobic region of membranes, while beta-sheeted peptides are predisposed to interact with polar groups and stay at the surface of lipid layers.

Summary Objective Pathological mineralization is induced by unbalance between pro- and anti-mineralization factors. In calcifying osteoarthritic joints, articular chondrocytes undergo terminal differentiation similar to that in growth plate cartilage and release matrix vesicles (MVs) responsible for hydroxyapatite (HA) or calcium pyrophosphate dihydrate (CPPD) deposition. Inorganic pyrophosphate (PP i ) is a likely source of inorganic phosphate (P i ) to sustain HA formation when hydrolyzed but also a potent inhibitor preventing apatite mineral deposition and growth. Moreover, an excess of PP i can lead to CPPD formation, a marker of pathological calcification in osteoarthritic joints. It was suggested that the P i /PP i ratio during biomineralization is a turning point between physiological and pathological mineralization. The aim of this work was to determine the conditions favoring either HA or CPPD formation initiated by MVs. Methods MVs were isolated from 17-day-old chicken embryo growth plate cartilages and subjected to mineralization in the presence of various P i /PP i ratios. The mineralization kinetics and the chemical composition of minerals were determined, respectively, by light scattering and infrared spectroscopy. Results The formation of HA is optimal when the P i /PP i molar ratio is above 140, but is completely inhibited when the ratio decreases below 70. The retardation of any mineral formation is maximal at P i /PP i ratio around 30. CPPD is exclusively produced by MVs when the ratio is below 6, but it is inhibited for the ratio exceeding 25. Conclusions Our findings are consistent with the P i /PP i ratio being a determinant factor leading to pathological mineralization or its inhibition.

We show that temperature is an important parameter for the sensitivity of saturation transfer difference (STD) spectroscopy. A decreased intensity of STD signals is observed for lactose binding to growth-regulatory galectin7 (p53-induced gene 1), as well as for nucleotide binding to annexin A6, when the temperature is increased from 281 to 298-310 K. Opposite temperature effects on STD intensity are observed for S-peptide binding to S-protein to reconstitute RNase S. However, the STD signals for tryptophan binding to downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM)are relatively unaffected between 281 and 298 K. The known kinetics of the binding of ATP by the uncoupling protein from brown adipose tissue mitochondria (UCP1) predicted an observable STD at 310 K, but rapid sample degradation limits the experiments to much lower temperatures. Temperature strongly influences the kinetics and affinity constant of various types of complex formation and in so doing influences the observed STD effects. Therefore, temperature can be exploited to facilitate the optimization of STD-based applications, and at the same time minimize the number of test samples. STD-based screening protocols to detect new target-specific compounds may yield a larger number of potential ligands if screened at various temperatures.

International audience; Our knowledge about mesenchymal stem cells has considerably grown in the last years. Since the proof of concept of the existence of such cells in the 70s by Friedenstein et al., a growing mass of reports were conducted for a better definition of these cells and for the reevaluation from the term “mesenchymal stem cells” to the term “mesenchymal stromal cells (MSCs).” Being more than a semantic shift, concepts behind this new terminology reveal the complexity and the heterogeneity of the cells grouped in MSC family especially as these cells are present in nearly all adult tissues. Recently, mesenchymal stromal cell antigen-1 (MSCA-1)/tissue nonspecific alkaline phosphatase (TNAP) was described as a new cell surface marker of MSCs from different tissues. The alkaline phosphatase activity of this protein could be involved in wide range of MSC features described below from cell differentiation to immunomodulatory properties, as well as occurrence of pathologies. The present review aims to decipher and summarize the role of TNAP in progenitor cells from different tissues focusing preferentially on brain, bone marrow, and adipose tissue.

International audience; A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min-1 mg-1 for PPi, to 56 ± 11 nmol min-1 mg-1 for AMP, to 79 ± 23 nmol min-1 mg-1 for beta-glycerophosphate and to 73 ± 15 nmol min-1 mg-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of β-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6–12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.

Correction for ‘Giant vesicles from rehydrated crude mixtures containing unexpected mixtures of amphiphiles formed under plausibly prebiotic conditions’ by Michele Fiore et al., Org. Biomol. Chem., 2017, 15, 4231–4240.

Conformational changes occurring in the catalytic cycle of the H+/K+-ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca2+, to induce the transition between the E1 and E1-P conformation of the H+/K+-ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of Pi from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.

International audience; Bone is a dynamic tissue that is constantly renewed by the coordinated action of two cell types, i.e., the bone-resorbing osteoclasts and the bone-forming osteoblasts. However, in some circumstances, bone regeneration exceeds bone self repair capacities. This is notably often the case after bone fractures, osteolytic bone tumor surgery, or osteonecrosis. In this regard, bone tissue engineering with autologous or allogenic mesenchymal stem cells (MSCs) is been widely developed. MSCs can be isolated from bone marrow or other tissues such as adipose tissue or umbilical cord, and can be implanted in bone defects with or without prior amplification and stimulation. However, the outcome of most pre-clinical studies remains relatively disappointing. A better understanding of the successive steps and molecular mechanisms involved in MSC-osteoblastic differentiation appears to be crucial to optimize MSC-bone therapy. In this review, we first present the important growth factors that stimulate osteoblastogenesis. Then we review the main transcription factors that modulate osteoblast differentiation, and the microRNAs (miRs) that inhibit their expression. Finally, we also discuss articles dealing with the use of these factors and miRs in the development of new bone MSC therapy strategies. We particularly focus on the studies using human MSCs, since significant differences exist between osteoblast differentiation mechanisms in humans and mice for instance.

International audience; Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.

International audience; The present review aims to systematically and critically analyze the current knowledge on phospholipases and their role in physiological and pathological mineralization undertaken by mineralization competent cells. Cellular lipid metabolism plays an important role in biological mineralization. The physiological mechanisms of mineralization are likely to take place in tissues other than in bones and teeth under specific pathological conditions. For instance, vascular calcification in arteries of patients with renal failure, diabetes mellitus or atherosclerosis recapitulates the mechanisms of bone formation. Osteoporosis-a bone resorbing disease-and rheumatoid arthritis originating from the inflammation in the synovium are also affected by cellular lipid metabolism. The focus is on the lipid metabolism due to the effects of dietary lipids on bone health. These and other phenomena indicate that phospholipases may participate in bone remodelling as evidenced by their expression in smooth muscle cells, in bone forming osteoblasts, chondrocytes and in bone resorbing osteoclasts. Among various enzymes involved, phospholipases A1 or A2, phospholipase C, phospholipase D, autotaxin and sphingomyelinase are engaged in membrane lipid remodelling during early stages of mineralization and cell maturation in mineralization-competent cells. Numerous experimental evidences suggested that phospholipases exert their action at various stages of mineralization by affecting intracellular signaling and cell differentiation. The lipid metabolites-such as arachidonic acid, lysophospholipids, and sphingosine-1-phosphate are involved in cell signaling and inflammation reactions. Phospholipases are also important members of the cellular machinery engaged in matrix vesicle (MV) biogenesis and exocytosis. They may favour mineral formation inside MVs, may catalyse MV membrane breakdown necessary for the release of mineral deposits into extracellular matrix (ECM), or participate in hydrolysis of ECM. The biological functions of phospholipases are discussed from the perspective of animal and cellular knockout models, as well as disease implications, development of potent inhibitors and therapeutic interventions.

Light-induced release of ADP and ATP from their respective caged nucleotides produced small distinct difference infrared spectra of creatine kinase (CK), indicating that ADP and ATP binding to CK promoted different structural alteration. The positive band at 1638-1640 cm-1 and the negative band at about 1650-1652 cm-1 on the reaction-induced infrared difference spectra in the amide I region were insensitive to the deuteration effects. They were assigned to the peptide backbone of the ADP/ATP-binding site. In addition Pi or ATP binding produced another positive band at 1657-1659 cm-1 corresponding to the C = O (amide I band) associated with the gamma-phosphate of ATP. This site was also affected when ADP was added, indicating coupling interactions between both sites. No additional structural changes were observed when creatine and ADP were added, suggesting that the creatine-binding site was uncoupled from the ADP-binding site. The infrared difference spectra of a transition-state-analog complex formed by the addition of ADP, creatine and NO3- (a planar-phosphate-mimicking group) lacked the 1657-1659-cm-1 band indicating that the binding site of gamma-phosphate within CK, was not affected. Infrared changes in the 1560-1590-cm-1 region suggested that carboxylate groups of Asp or Glu were involved in the binding of Pi, ADP and ATP.

SPECIFIC AIMSWe tested the hypothesis that annexin VI (AnxVI), a member of a family of Ca2+ and lipid binding proteins, can interact with membranes in a Ca2+-independent manner and behave as a membrane integral protein. For this purpose, we investigated the consequences of AnxVI pH-dependent binding to lipid membranes, such as the formation of ion channels by the annexin molecules and pH-induced conformational changes of AnxVI, to understand some functional aspects of the protein in vivo, particularly under pathological conditions, connected with local fluctuations of pH.PRINCIPAL FINDINGS1. The binding of AnxVI to phospholipids in a Ca2+-insensitive, pH-dependent manner constitutes an alternative to the Ca2+ bridging mechanism of interaction of annexins with membranesThe Ca2+-independent binding of AnxVI to asolectin liposomes is stimulated by acidic pH; half-maximal binding occurred at pH 5.3. The lipid composition of liposomes is not crucial for AnxVI–lipid interactions at low pH. The binding at lower ...

5 páginas, 4 figuras -- PAGS nros. 447-451, Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested, This work was supported in part by a Spain–France bilateral grant to R.B./J.C. funded by the Spanish Ministry of Science (HF2005-0276) and Égide (Picasso N 10715SA), by an exchange program sponsored by the Polish Academy of Sciences (2009-2010/5) and Fundação para a Ciência e a Tecnologia to S.P./P.G, and by grant N401 049 32/1143 to J.B-P. from the Polish Ministry of Science and Higher Education

It is well established that the octameric mitochondrial form of creatine kinase (mtCK) binds to the outer face of the inner mitochondrial membrane mainly via electrostatic interactions with cardiolipin (CL). However, little is known about the consequences of these interactions on membrane and protein levels. Brewster angle microscopy investigations provide, for the first time to our knowledge, images indicating that mtCK binding induced cluster formation on CL monolayers. The thickness of the clusters (10-12 nm) corresponds to the theoretical height of the mtCK-CL complex. Protein insertion into a condensed CL film, together with monolayer stabilization after protein addition, was observed by means of differential capacity measurements. Polarization modulation infrared reflection-absorption spectroscopy showed that the mean orientation of alpha-helices within the protein shifted upon CL binding from 30 degrees to 45 degrees with respect to the interface plane, demonstrating protein domain movements. A comparison of data obtained with CL and phosphatidylcholine/phosphatidylethanolamine/CL (2:1:1) monolayers indicates that mtCK is able to selectively recruit CL molecules within the mixed monolayer, consolidating and changing the morphology of the interfacial film. Therefore, CL-rich domains induced by mtCK binding could modulate mitochondrial inner membrane morphology into a raft-like organization and influence essential steps of mitochondria-mediated apoptosis.

Lansoprazole, a known H(+)/K(+)-ATPase inhibitor, is currently used as a therapeutical option for the initial treatment of gastroesophageal reflux disease. Recently, lansoprazole has been found to be an inhibitor of cytosolic PHOSPHO1 (a phosphatase which hydrolyses phosphocholine and phosphoethanolamine), providing a possible therapeutical target to cure pathological mineralization. Since PHOSPHO1 is present inside matrix vesicles, we tested the effect of lansoprazole on matrix vesicles containing several key enzymes for the mineralization process including tissue-nonspecific alkaline phosphatase. We found that lansoprazole can inhibit in an uncompetitive manner tissue-nonspecific alkaline phosphatase. A K(i) value of 1.74 +/- 0.12 mM has been determined for the inhibition of tissue-nonspecific alkaline phosphatase by lansoprazole. Lansoprazole, currently used for treating gastroesophageal disease, by inhibiting PHOSPHO1 and tissue-nonspecific alkaline phosphatase could prevent hydroxyapatite-deposition disease and could serve as an adjunct treatment for osteoarthritis.