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2812 Matrix Vesicles: Structure, Composition, Formation and Function in Ca

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[Frontiers in Bioscience 16, 2812-2902, June 1, 2011] Matrix vesicles: structure, composition, formation and function in calcification Roy E. Wuthier Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Morphology of matrix vesicles (MVs) 3.1. Conventional transmission electron microscopy 3.2. Cryofixation, freeze-substitution electron microscopy 3.3. Freeze-fracture studies 4. Isolation of MVs 4.1. Crude collagenase digestion methods 4.2. Non-collagenase dependent methods 4.3. Cell culture methods 4.4. Modified collagenase digestion methods 4.5. Other isolation methods 5. MV proteins 5.1. Early SDS-PAGE studies 5.2. Isolation and identification of major MV proteins 5.3. Sequential extraction, separation and characterization of major MV proteins 5.4. Proteomic characterization of MV proteins 6. MV-associated extracellular matrix proteins 6.1. Type VI collagen 6.2. Type X collagen 6.3. Proteoglycan link protein and aggrecan core protein 6.4. Fibrillin-1 and fibrillin-2 7. MV annexins – acidic phospholipid-dependent ca2+-binding proteins 7.1. Annexin A5 7.2. Annexin A6 7.3. Annexin A2 7.4. Annexin A1 7.5. Annexin A11 and Annexin A4 8. MV enzymes 8.1. Tissue-nonspecific alkaline phosphatase(TNAP) 8.1.1. Molecular structure 8.1.2. Amino acid sequence 8.1.3. 3-D structure 8.1.4. Disposition in the MV membrane 8.1.5. Catalytic properties 8.1.6. Collagen-binding properties 8.2. Nucleotide pyrophosphate phosphodiesterase (NPP1, PC1) 8.3. PHOSPHO-1 (Phosphoethanolamine/Phosphocholine phosphatase 8.4. Acid phosphatase 8.5. Proteases and peptidases 8.6. Lactate dehydrogenase and other glycolytic enzymes 8.7. Carbonic anhydrase 8.8. Phospholipases 8.8.1. Phospholipase A (PLA) 8.8.2. Phospholipase C (PLC) 8.8.3. Lysophospholipase C (LPLC) 8.8.4. Sphingomyelinase (Smase) 8.8.5. Other MV phospholipases 8.9. Phosphatidylserine synthases 8.10. Phospholipid flippases and scramblases 9. MV transporters and surface receptors 9.1. Cell-surface receptors 9.2. ATP-driven ion pumps and transporters 9.3. Channel proteins and solute-carrier transporters 9.4. Ca2+ channel proteins 2812 Function of matrix vesicles in calcification 9.4.1. L-type Ca2+ channels in growth plate chondrocytes 9.4.2. Annexins as Ca2+ channels 9.5. Inorganic P (Pi) transporters 9.5.1. Na+-dependent Pi transporters 9.5.2. Na+-independent Pi transporters 10. MV regulatory proteins 10.1. Syntenin 10.2. Gi Protein, alpha-2 10.3. Trp/Trp mono-oxygenase activation protein, etc. 11. MV cytoskeletal proteins 11.1. Actin 11.2. Filamen B, beta 11.3. Actinin 11.4. Tubulin 11.5. Radixin 11.6. Gelsolin and proteins with gelsolin/villin domains 11.7. Plastins 12. MV lipids 12.1. Phospholipids 12.1.1. Fatty acid composition 12.1.2. Physical form of MV phospholipids 12.1.3. Topological distribution of MV phospholipids 12.1.4. Ca2+-binding properties of MV phospholipids 12.2. Nonpolar lipids 12.3. Glycolipids 13. MV electrolytes 13.1. Chemical analyses of intracellular electrolytes in growth plate chondrocytes 13.2. Chemical analyses of electrolytes in isolated MVs 13.3. Chemical analyses of electrolytes in the extracellular fluid and blood plasma 13.4. Ultrastructural analyses of Ca2+ and P in cells and MVs 13.5. Chemical analyses of micro-electrolytes in MVs 13.6. Physico-chemical properties of MV mineral forms 13.6.1. X-ray diffraction 13.6.2. FT-IR and FT-Raman analyses 13.7. Solubility properties of nascent MV mineral 14. Formation of MVs 14.1. Electron microscopic studies 14.2. Biochemical studies 14.2.1. Phospholipid metabolic studies 14.2.2. Cell culture studies 14.3. Role of cellular Ca2+ and Pi metabolism in MV formation 14.3.1. Histology of cellular Ca2+ metabolism during MV formation 14.3.2. Confocal imaging of Ca2+ in living growth plate chondrocytes 14.3.3. Biochemistry of mitochondrial Ca2+ metabolism 14.4. Growth plate cellular Pi metabolism 14.4.1. Chondrocyte mitochondrial PIC Pi-transporter 14.4.2. Chondrocyte plasma membrane Pi transporters 14.4.3. Chondrocyte mitochondrial permeability transition (MPT) pore 14.5. Growth plate cellular H+ (pH) metabolism 14.5.1. Confocal imaging of H+ (pH) in living growth plate chondrocytes 15. Kinetics of mineral formation by MVs 15.1. Differing views of MV mineral formation 15.2. Radio-isotope analyses of the kinetics of MV Ca2+ and Pi accumulation 15.2.1. Stage 1 – Initial exchange 15.2.2. Stage 2 – Lag period 15.2.3. Stage 3 – Induction period 15.2.4. Stage 4 – Rapid acquisition period 15.2.5. Stage 5 – Plateau period 16. The nucleation core: The driving force in MV Ca2+ and Pi accumulation 16.1. Discovery of the nucleation core (NC) 16.2. Isolation of the NC 16.3. Chemical characterization of the NC 2813 Function of matrix vesicles in calcification 16.3.1. Electrophoretic analysis of proteins 16.3.2. Chromatographic analysis of lipids 16.4. Physical characterization of the NC 16.4.1. Transmission electron microscopy (TEM) 16.4.2. FT-IR analysis 16.4.3. 31P-NMR analysis 16.4.4. pH sensitivity 16.5. Summary of NC structure and composition 17. Reconstitution of the NC: role of PS-Ca2+-Pi complex (PS-CPLX) in MV crystal nucleation 17.1. Reconstitution of the nucleational core 17.2. Mathematical analysis of the kinetics of mineral formation 17.3. The physical and nucleational properties of pure PS-CPLX 17.4. Requirements for PS-CPLX formation: pH tolerance 17.5. Molecular rearrangements during PS-CPLX formation 17.6. Effect of Mg2+ incorporation on nucleation activity of PS-CPLX 17.7. Role of AnxA5 in MV crystal nucleation 17.8. Mechanism of MV nucleation of OCP 18. Modeling of MV using synthetic unilamellar liposomes 18.1. Effects of Ca2+ ionophores on Ca2+ uptake 18.2. Minimal effects of AnxA5 on 45Ca2+ uptake 18.3. Effects of detergents and phospholipase A2 18.4. Proteoliposomal MV models by other groups 19. Regulation of MV Calcification 19.1. Factors that primarily effect nucleation 19.2. Effects of phospholipid composition 19.3. Stimulatory effects of AnxA5 on MV nucleation 19.4. Effect of non-apatitic electrolytes 19.4.1. Effects of Mg2+ 19.4.2. Effects of Zn2+ 19.4.3. Effects of PPi 19.5. Inhibitory effects of proteoglycans 19.6. Stimulatory effects of type II and X collagens 19.7. Effects of non-collagenous bone-related proteins 20. Further evaluation of the roles of key MV proteins 20.1. Tissue-nonspecific alkaline phosphatase (TNAP) 20.1.1. Role of TNAP in PPi hydrolysis 20.1.2. Additional roles of TNAP 20.1.3. Improperly assigned roles of TNAP 20.2. Annexin A5 (AnxA5) 20.2.1. Questionable role in Ca2+ entrance into MVs 20.2.2. Activation of the nucleational core 20.2.3. Does AnxA5 have a physiological function? 20.2.4. Effects of double knockout of AnxA5 and AnxA6 on expression of other genes 20.2.5. Possible substitution of PS-receptor (Ptdsr) for AnxA5 20.2.6. What would be the effects of expression of defective AnxA5 mutants? 20.2.7. Is the need for AnxA5 stress-dependent? 20.3. PS Synthase 20.3.1. PS synthesis is not high-energy dependent, but requires Ca2+ 20.3.2. PSS1 and PSS2 gene knockouts 20.4. Phospholipase A and C activities 20.4.1. Membrane lysis and Ca2+ access to nucleational core 20.4.2. Role in the egress of mineral from the vesicle lumen 20.4.3. Salvage of phospholipid P for mineral formation 20.5. PHOSPHO-1 20.5.1. Salvage of Pi and choline+ from phospholipid breakdown 20.5.2. Inhibition of PHOSPHO1 activity 20.5.3. PHOSPHO1 gene knockout 20.5.4. Co-modulation of TNAP and PHOSPHO1 gene expression 20.5.5. Effect on MV mineral formation 21. Perspectives 22. Acknowledgements 23. References 2814 Function of matrix vesicles in calcification 1. ABSTRACT however, much of the subsequent research utilized developing bones of hybrid, broiler-strain chickens because Matrix vesicles (MVs) induce calcification of the exceptional amounts of material readily available at during endochondral bone formation. Experimental very low cost. This has enabled direct biochemical analysis methods for structural, compositional, and functional of key components critical for elucidation of this highly analysis of MVs are reviewed. MV proteins, enzymes, complex system. Others, in order to overcome the problem receptors, transporters, regulators, lipids and electrolytes of insufficient tissue mass, have resorted to the use of are detailed. MV formation is considered from both rachitic rats. However, none of these experimental structural and biochemical perspectives. Confocal imaging approaches is necessarily ideal. In comparison to the of Ca2+ and H+ were used to depict how living mouse, the human skeleton is massive, over one-thousand chondrocytes form MVs. Biochemical studies revealed that times larger, and it lasts over one-hundred times longer. coordinated mitochondrial Ca2+ and Pi metabolism produce Thus, it is doubtful that mineral formation in a diminutive MVs containing a nucleational complex (NC) of vertebrate like the mouse is entirely analogous to that in amorphous calcium phosphate, phosphatidylserine and humans. On the other hand, the extreme demands for rapid annexin A5 – all critical to the mechanism of mineral growth and mineralization of bone in the broiler-strain nucleation. Reconstitution of the NC and modeling with chicken may well require specialized features that are not unilamellar vesicles reveal how the NC transforms into required for skeletal development of the mouse – or the octacalcium phosphate, regulated by Mg2+, Zn2+ and human – except perhaps during the pubertal growth spurt. annexin A5.
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  • Genetic, Cytogenetic and Physical Refinement of the Autosomal Recessive CMT Linked to 5Q31ð Q33: Exclusion of Candidate Genes I

    Genetic, Cytogenetic and Physical Refinement of the Autosomal Recessive CMT Linked to 5Q31ð Q33: Exclusion of Candidate Genes I

    European Journal of Human Genetics (1999) 7, 849–859 © 1999 Stockton Press All rights reserved 1018–4813/99 $15.00 t http://www.stockton-press.co.uk/ejhg ARTICLE Genetic, cytogenetic and physical refinement of the autosomal recessive CMT linked to 5q31–q33: exclusion of candidate genes including EGR1 Ang`ele Guilbot1, Nicole Ravis´e1, Ahmed Bouhouche6, Philippe Coullin4, Nazha Birouk6, Thierry Maisonobe3, Thierry Kuntzer7, Christophe Vial8, Djamel Grid5, Alexis Brice1,2 and Eric LeGuern1,2 1INSERM U289, 2F´ed´eration de Neurologie and 3Laboratoire de Neuropathologie R Escourolle, Hˆopital de la Salpˆetri`ere, Paris 4Laboratoire de cytog´en´etique, Villejuif 5G´en´ethon, Evry, France 6Service de Neurologie, Hˆopital des Sp´ecialit´es, Rabat, Morocco 7Service de Neurologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 8Service D’EMG et de pathologie neuromusculaire, Hˆopital neurologique Pierre Wertheimer, Lyon, France Charcot-Marie-Tooth disease is an heterogeneous group of inherited peripheral motor and sensory neuropathies with several modes of inheritance: autosomal dominant, X-linked and autosomal recessive. By homozygosity mapping, we have identified, in the 5q23–q33 region, a third locus responsible for an autosomal recessive form of demyelinating CMT. Haplotype reconstruction and determination of the minimal region of homozygosity restricted the candidate region to a 4 cM interval. A physical map of the candidate region was established by screening YACs for microsatellites used for genetic analysis. Combined genetic, cytogenetic and physical mapping restricted the locus to a less than 2 Mb interval on chromosome 5q32. Seventeen consanguineous families with demyelinating ARCMT of various origins were screened for linkage to 5q31–q33.
  • Methylome and Transcriptome Maps of Human Visceral and Subcutaneous

    Methylome and Transcriptome Maps of Human Visceral and Subcutaneous

    www.nature.com/scientificreports OPEN Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal Received: 9 April 2019 Accepted: 11 June 2019 key epigenetic diferences at Published: xx xx xxxx developmental genes Stephen T. Bradford1,2,3, Shalima S. Nair1,3, Aaron L. Statham1, Susan J. van Dijk2, Timothy J. Peters 1,3,4, Firoz Anwar 2, Hugh J. French 1, Julius Z. H. von Martels1, Brodie Sutclife2, Madhavi P. Maddugoda1, Michelle Peranec1, Hilal Varinli1,2,5, Rosanna Arnoldy1, Michael Buckley1,4, Jason P. Ross2, Elena Zotenko1,3, Jenny Z. Song1, Clare Stirzaker1,3, Denis C. Bauer2, Wenjia Qu1, Michael M. Swarbrick6, Helen L. Lutgers1,7, Reginald V. Lord8, Katherine Samaras9,10, Peter L. Molloy 2 & Susan J. Clark 1,3 Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional diferences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic diferences and their contribution to cell type and depot-specifc function. We found that DNA methylomes were notably distinct between diferent adipocyte depots and were associated with diferential gene expression within pathways fundamental to adipocyte function. Most striking diferential methylation was found at transcription factor and developmental genes. Our fndings highlight the importance of developmental origins in the function of diferent fat depots.
  • Changes in the Cytosolic Proteome of Aedes Malpighian Tubules

    Changes in the Cytosolic Proteome of Aedes Malpighian Tubules

    329 The Journal of Experimental Biology 212, 329-340 Published by The Company of Biologists 2009 doi:10.1242/jeb.024646 Research article Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules Klaus W. Beyenbach1,*, Sabine Baumgart2, Kenneth Lau1, Peter M. Piermarini1 and Sheng Zhang2 1Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca, NY 14853, USA and 2Proteomics and Mass Spectrometry Core Facility, 143 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA *Author for correspondence (e-mail: [email protected]) Accepted 6 November 2008 Summary Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10–7 mol l–1). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of ±1.5 were robotically picked for in- gel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK- III reduces the cytosolic presence of subunits A and B of the V-type H+ ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca2+-binding protein regucalcin/SMP30 and actin-depolymerizing factor.