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Symmetry Breaking of Phospholipids

Michele Fiore, René Buchet
2020 Symmetry  
Either stereo reactants or stereo catalysis from achiral or chiral molecules are a prerequisite to obtain pure enantiomeric lipid derivatives. We reviewed a few plausibly organic syntheses of phospholipids under prebiotic conditions with special attention paid to the starting materials as pro-chiral dihydroxyacetone and dihydroxyacetone phosphate (DHAP), which are the key molecules to break symmetry in phospholipids. The advantages of homochiral membranes compared to those of heterochiral
more » ... nes were analysed in terms of specific recognition, optimal functions of enzymes, membrane fluidity and topological packing. All biological membranes contain enantiomerically pure lipids in modern bacteria, eukarya and archaea. The contemporary archaea, comprising of methanogens, halobacteria and thermoacidophiles, are living under extreme conditions reminiscent of primitive environment and may indicate the origin of one ancient evolution path of lipid biosynthesis. The analysis of the known lipid metabolism reveals that all modern cells including archaea synthetize enantiomerically pure lipid precursors from prochiral DHAP. Sn-glycerol-1-phosphate dehydrogenase (G1PDH), usually found in archaea, catalyses the formation of sn-glycerol-1-phosphate (G1P), while sn-glycerol-3-phosphate dehydrogenase (G3PDH) catalyses the formation of sn-glycerol-3-phosphate (G3P) in bacteria and eukarya. The selective enzymatic activity seems to be the main strategy that evolution retained to obtain enantiomerically pure lipids. The occurrence of two genes encoding for G1PDH and G3PDH served to build up an evolutionary tree being the basis of our hypothesis article focusing on the evolution of these two genes. Gene encoding for G3PDH in eukarya may originate from G3PDH gene found in rare archaea indicating that archaea appeared earlier in the evolutionary tree than eukarya. Archaea and bacteria evolved probably separately, due to their distinct respective genes coding for G1PDH and G3PDH. We propose that prochiral DHAP is an essential molecule since it provides a convergent link between G1DPH and G3PDH. The synthesis of enantiopure phospholipids from DHAP appeared probably firstly in the presence of chemical catalysts, before being catalysed by enzymes which were the products of later Darwinian selection. The enzymes were probably selected for their efficient catalytic activities during evolution from large libraries of vesicles containing amino acids, carbohydrates, nucleic acids, lipids, and meteorite components that induced symmetry imbalance.
doi:10.3390/sym12091488 fatcat:fefijxn5jrgvrjd2vmpgyj4kfi

Phosphorylation-dependent phospholipase D activity of matrix vesicles

Marcin Balcerzak, Slawomir Pikula, Rene Buchet
2006 FEBS Letters  
A progressive hydrolysis of phospholipids was observed during the mineralization process mediated by extracellular matrix vesicles. Increasing levels of different hydrolysis products revealed phospholipase A and D activities. The importance of these enzymes for the mineralization process lies in a high rate of hydrolysis of neutral phospholipids and lower rate of degradation of anionic phospholipids, which may favor mineral formation in vesicular membrane and membrane breakdown necessary for
more » ... release of mineral deposits into extracellular matrix. In this report, we focus on the phosphorylation-dependent phospholipase D activity during mineral formation initiated by chicken embryo matrix vesicles.
doi:10.1016/j.febslet.2006.09.018 pmid:16997299 fatcat:km4keeoasbczzdxoddvxvlwmpu

A novel retinoid binding property of human annexin A6

Marcin Balcerzak, Joanna Bandorowicz-Pikula, Rene Buchet, Slawomir Pikula
2006 FEBS Letters  
Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the
more » ... is of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.
doi:10.1016/j.febslet.2006.04.052 pmid:16674946 fatcat:qdnpxnkzhjcbjg5pqbkz7tcy64

Direct determination of phospholipase D activity by infrared spectroscopy

Le Duy Do, René Buchet, Slawomir Pikula, Abdelkarim Abousalham, Saida Mebarek
2012 Analytical Biochemistry  
To determine phospholipase D (PLD) activity an infrared spectroscopy assay was developed, based on the phosphate vibrational mode of the phospholipid substrates of the enzyme such as dimyristoylphophatidylcholine (DMPC), lysophosphatidylglycerol (lysoPG) dimyristoylethanolamine (DMPE), and lysophosphatidylserine (lysoPS). Characteristic vibrational bands were located at 1230, 1226, 1221 and 1218 cm -1 , respectively, and served to monitor the hydrolysis of phospholipids. The appearance of the
more » ... osphate vibrational band of phosphatidate at 1130 cm -1 , served to monitor the amount of byproduct of the hydrolytic cleavage of phospholipids by PLD. In situ measurements could be performed within less than 20 min, using 2-40 mM DMPC and at least 5-10 ng of S. chromofucus PLD having specific activity of 30 nmol min -1 µg -1 ( corresponding to 150-300 pmol hydrolysed DMPC per minute) at pH 8.0 in the presence of 10 mM Ca 2+ . The feasibility of the infrared assay using lysoPG, DMPE and lysoPS was also demonstrated, indicating that various natural phospholipids could be employed as substrates to measure the PLD activity. Reproducible apparent maximum velocities (V max ) were also determined. The direct infrared assay could be used as a possible screening tool to find specific PLD inhibitors. Le Duy Do Is a recipient of the fellowship from the International PhD program in Neurobiology. This project is supported by POLONIUM (Project N° 27727TA), PICS (Project N° 5096) and the Nencki Institute of Experimental Biology.
doi:10.1016/j.ab.2012.07.017 pmid:22842398 fatcat:afvbiksywnca5iedwz3hwuf3qu

Racemic Phospholipids for Origin of Life Studies

Emiliano Altamura, Arnaud Comte, Alice D'Onofrio, Charlotte Roussillon, Dimitri Fayolle, René Buchet, Fabio Mavelli, Pasquale Stano, Michele Fiore, Peter Strazewski
2020 Symmetry  
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
more » ... lketal (α,β-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%.
doi:10.3390/sym12071108 fatcat:rjoj6mkypbe5zliwyvb5lmnbg4

Mitochondrial Creatine Kinase Binding to Phospholipid Monolayers Induces Cardiolipin Segregation

Ofelia Maniti, Marie-France Lecompte, Olivier Marcillat, Bernard Desbat, René Buchet, Christian Vial, Thierry Granjon
2009 Biophysical Journal  
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
more » ... usters (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 a-helices within the protein shifted upon CL binding from 30 to 45 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.
doi:10.1016/j.bpj.2008.12.3911 pmid:19289067 pmcid:PMC2907684 fatcat:abbld5vgpredna4lhedzcjev6y

Peroxidase activity of annexin 1 from Arabidopsis thaliana

Karolina M. Gorecka, Dorota Konopka-Postupolska, Jacek Hennig, Rene Buchet, Slawomir Pikula
2005 Biochemical and Biophysical Research Communications - BBRC  
On the basis of earlier reports suggesting that annexin A1 from Arabidopsis thaliana (AnnAt1) participates in limiting the excessive levels of reactive oxygen species during oxidative burst in plants, we examined the sensitivity of recombinant AnnAt1 to hydrogen peroxide and its peroxidase activity. Purified recombinant protein remains mostly a-helical and binds to lipids in a calcium-dependent manner. Upon oxidation recombinant AnnAt1 exhibits a tendency to form dimers in vitro. AnnAt1 is also
more » ... sensitive to the presence of reducing agents, suggesting that AnnAt1 is a redox sensor in plant cells. Moreover, using two independent methods we found that AnnAt1 displayed peroxidase activity which is probably related to the presence of a heme-binding domain within AnnAt1, as present in other peroxidases. Indeed, site-directed mutagenesis within this domain resulted in a complete abrogation of the activity of AnnAt1. Furthermore, this activity was found to be sensitive to the phosphorylation state of the protein.
doi:10.1016/j.bbrc.2005.08.181 pmid:16153598 fatcat:kxq63j6ovvgwver257oeha6ku4

Distinct structure and activity recoveries reveal differences in metal binding between mammalian andEscherichia colialkaline phosphatases

Le Zhang, René Buchet, Gérard Azzar
2005 Biochemical Journal  
The amino acids involved in the coordination of two Zn 2+ ions and one Mg 2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although
more » ... sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries. After mild chelation inactive apo-EAP was monomeric under mild denaturing conditions, whereas inactive apo-BIAP remained dimeric, indicating that the monomer-monomer contact was stronger in the mammalian enzyme. Dimeric apo-EAP (0.45 µM, corresponding to 4 units/ml) recovered approx. 80 % of its initial activity after 3 min incubation in an optimal recovery medium containing 5 µM Zn 2+ and 5 mM Mg 2+ , whereas dimeric apo-BIAP (0.016 µM, corresponding to 4 units/ml) recovered 80 % of its native activity after 6 h incubation in an optimal recovery medium containing 0.5 µM Zn 2+ and 5 mM Mg 2+ . Small and different secondary structure changes were also observed during activity recoveries of apo-BIAP and apo-EAP, which were not in parallel with the activity recoveries, suggesting that distinct and subtle structural changes are required for their optimal activity recoveries.
doi:10.1042/bj20050509 pmid:16086666 pmcid:PMC1316277 fatcat:s56s7inzfvgn5piceejky2ec7i

Multiple Functions of MSCA-1/TNAP in Adult Mesenchymal Progenitor/Stromal Cells

David Estève, Jean Galitzky, Anne Bouloumié, Caroline Fonta, René Buchet, David Magne
2016 Stem Cells International  
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
more » ... ouped 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.
doi:10.1155/2016/1815982 pmid:26839555 pmcid:PMC4709781 fatcat:2pdetrpzmjacrlnegvxvz7vrhu

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

Zhongyuan Ren, Le Duy Do, Géraldine Bechkoff, Saida Mebarek, Nermin Keloglu, Saandia Ahamada, Saurabh Meena, David Magne, Slawomir Pikula, Yuqing Wu, René Buchet, Elena Aikawa
2015 PLoS ONE  
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
more » ... neralization 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 levamisolea TNAP inhibitorserved to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC 50 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.
doi:10.1371/journal.pone.0120087 pmid:25785438 pmcid:PMC4364917 fatcat:yqqpdc7oprgalbwydhnqiutdxe

GTP-Induced Membrane Binding and Ion Channel Activity of Annexin VI: Is Annexin VI a GTP Biosensor?

Aneta Kirilenko, Marcin Golczak, Slawomir Pikula, Rene Buchet, Joanna Bandorowicz-Pikula
2002 Biophysical Journal  
Annexin VI (AnxVI) formed ion channels in planar lipid bilayers that were induced by the addition of millimolar guanosine 5Ј-triphosphate (GTP) at pH 7.4 and that were not accompanied by a penetration of the protein into the membrane hydrophobic region. GTP-influenced interactions of AnxVI with Ca 2ϩ /liposomes produced small structural alterations as revealed by circular dichroism and infrared spectroscopies. Guanosine 5Ј-3-O-(thio)-triphosphate (GTP␥S) binding to AnxVI, promoted by the
more » ... lease of GTP␥S from GTP␥S[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged-GTP␥S), affected three to four amino acid residues of AnxVI in the presence of Ca 2ϩ /liposomes, while about eight or nine amino acid residues were altered in their absence. This suggested that the nucleotide-binding site overlapped the lipid-binding domain of AnxVI. The binding of the fluorescent GTP analog, 2Ј-(or 3Ј)-O-(2,4,6-trinitrophenyl)guanosine 5Ј-triphosphate (TNP-GTP) to AnxVI was optimal in the presence of Ca 2ϩ /liposomes, with a dissociation constant (K d ) of 1 M and stoichiometry of 1. TNP-GTP promoted fluorescence resonance energy transfer from tryptophan residues to the nucleotide. Ion conductance and fluorescence measurements of the C-and N-terminal fragments of AnxVI indicated distinct GTP-binding properties, suggesting that the existence of the GTP-induced ion channel activity of AnxVI is associated with the flexibility of the two halves of the protein. Such structural flexibility could contribute to a molecular mechanism of AnxVI acting as a GTP biosensor. MATERIALS AND METHODS Materials GTP␥S[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged-GTP␥S) and 2Ј-(or 3Ј)-O-(2,4,6-trinitrophenyl)guanosine 5Ј-triphosphate (TNP-GTP) were
doi:10.1016/s0006-3495(02)75614-2 pmid:11964259 pmcid:PMC1302061 fatcat:4ea3wa6myzagxla5hl26rjv2ge

Emerging views on the structure and dynamics of the Ca2+-ATPase in sarcoplasmic reticulum

Anthony N. Martonosi, Istvan Jona, Elek Molnar, Norbert W. Seidler, Rene Buchet, Sandor Varga
1990 FEBS Letters  
The ATP-dependent Caz' transport in sarcoplasmic reticulum involves transitions between several structural states of the CaZ*-ATPase, that occur without major changes in the secondary structure. The rates ofthese transitions are modulated by the lipid enviroIl]~ent and by interactions between ATPase molecules. Although the Ca*+-ATPdse restricts the rotational mobility of a population of lipids, there is no evidence f&r specific interaction of the CazC-ATPase with phospholipids. Fluorescence
more » ... rization and energy transfer (FET) studies. using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca ZC-ATPase in which the binding sites of Ca*+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of'rigid' and 'flexible' regions within the molecule characterized. by different degrees of thermally induced structural fluctuations.
doi:10.1016/0014-5793(90)81287-x pmid:2143486 fatcat:brbx5o3vrndg3aysaormt6hqqq

Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments

Dimitri Fayolle, Emiliano Altamura, Alice D'Onofrio, Warren Madanamothoo, Bernard Fenet, Fabio Mavelli, René Buchet, Pasquale Stano, Michele Fiore, Peter Strazewski
2017 Scientific Reports  
et al.. Crude phosphorylation mixtures containing racemic lipid amphiphiles self-assemble to give stable primitive compartments. 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
more » ... 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 watersoluble probes, as evidenced by confocal microscopy and flow cytometry. Chemical reaction sideproducts (identified by IR and MS and quantified by 1 H 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. The spontaneous supramolecular self-assembly of amphiphiles to give vesicles is a powerful thermodynamic drive for the emergence of primitive cell-like compartments on the early Earth 1-7 . Fatty acid vesicles are plausible models of primitive cells 8,9 but contemporary cell membranes are based on mixtures of phospholipids, glycolipids and proteins. This elicits the questions about the stepwise transition from very early achiral or racemic amphiphiles to enantiopure glycerophospholipids. It has been argued that the complexification and the compositional evolution of primitive membranes was subjected not only to chemical rules (which chemical transformation is possible?) but also to a sort of supramolecular selection based on the 'performance' of the resulting membranes and whole vesicles -e.g., low critical aggregation (micelle, vesicle) concentration, membrane permeability, capture and retention of non-lipidic organic molecules, membrane growth and vesicle division upon the addition of membrane components, vesicle stability at high magnesium and calcium ions concentrations 8, 9 . Although chemistry suggests several plausible pathways for the stepwise transformation of simple amphiphiles into complex lipids 10-15 , the experimental verifications of the existence, stability, and properties of vesicles composed of plausibly primitive amphiphile mixtures are still limited 9,16-21 . Here we report on primitive membrane systems that originate from crude lipid phosphorylation mixtures. Our starting point is a non-phosphorylated diacylglycerol racemate. Glycerol is a reduced formose C 3 reaction Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SciEnTific REPORTS | (2017) 7:18106 |
doi:10.1038/s41598-017-18053-y pmid:29273739 pmcid:PMC5741756 fatcat:comozlcmwzamtpsgeth63nmrpy

Acyl chain composition determines cardiolipin clustering induced by mitochondrial creatine kinase binding to monolayers

Ofelia Maniti, Mouhedine Cheniour, Marie-France Lecompte, Olivier Marcillat, René Buchet, Christian Vial, Thierry Granjon
2011 Biochimica et Biophysica Acta - Biomembranes  
It has been recently shown that mitochondrial creatine kinase (mtCK) organizes mitochondrial model membrane by modulating the state and fluidity of lipids and by promoting the formation of proteincardiolipin clusters. This report shows, using Brewster angle microscopy, that such clustering is largely dependent on the acyl chain composition of phospholipids. Indeed, mtCK-cardiolipin domains were observed not only with unsaturated cardiolipins, but also with the cardiolipin precursor
more » ... lycerol. On the other hand, in the case of saturated dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin, mtCK was homogeneously distributed underneath the monolayer. However, an overall decrease in membrane fluidity was indicated by infrared spectroscopy as well as by extrinsic fluorescence spectroscopy using Laurdan as a fluorescent probe, both for tetramyristoylcardiolipin and bovine heart cardiolipin containing liposomes. The binding mechanism implicated the insertion of protein segments into monolayers, as evidenced from alternative current polarography, regardless of the chain unsaturation for the phosphatidylglycerols and cardiolipins tested. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m e m
doi:10.1016/j.bbamem.2011.01.005 pmid:21256109 fatcat:un7xemsh3be7hgb64vixwpuz6i

Src and ROCK Kinases Differentially Regulate Mineralization of Human Osteosarcoma Saos-2 Cells

Agnieszka Strzelecka-Kiliszek, Marta Romiszewska, Lukasz Bozycki, Saida Mebarek, Joanna Bandorowicz-Pikula, Rene Buchet, Slawomir Pikula
2019 International Journal of Molecular Sciences  
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
more » ... ciated coiled-coil (ROCK) kinases, which are involved in vesicular transport, may also regulate the mineralization process. Upon stimulation in osteogenic medium containing 50 μg/mL of ascorbic acid (AA) and 7.5 mM of β-glycerophosphate (β-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.
doi:10.3390/ijms20122872 pmid:31212828 pmcid:PMC6628028 fatcat:mfmnz64i45h57fiyhcvzw2xtou
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