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1.
Cell Mol Life Sci ; 70(2): 309-33, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22940918

ABSTRACT

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and ß we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin ß through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin ß, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.


Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Extracellular Matrix Proteins/metabolism , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Metalloproteases/antagonists & inhibitors , Metalloproteases/metabolism , ADAM10 Protein , Amino Acid Sequence , Animals , Caco-2 Cells , Cell Line , Cystatin C/metabolism , Cytokines/metabolism , Elafin/metabolism , HEK293 Cells , Humans , Mice , Mice, Knockout , alpha-2-HS-Glycoprotein/metabolism
2.
Macromol Biosci ; 13(2): 203-14, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23239639

ABSTRACT

Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulins/chemistry , Maleimides/chemistry , Polymethacrylic Acids/chemistry , Receptors, Immunologic/immunology , Binding Sites , Cell Line , Electrophoresis, Polyacrylamide Gel , Humans , Immunoglobulins/metabolism , Polymethacrylic Acids/chemical synthesis , Receptor for Advanced Glycation End Products , Receptors, Immunologic/metabolism
3.
J Control Release ; 163(2): 170-7, 2012 Oct 28.
Article in English | MEDLINE | ID: mdl-22981565

ABSTRACT

The successful non-invasive treatment of diseases associated with the central nervous system (CNS) is generally limited by poor brain permeability of various developed drugs. The blood-brain barrier (BBB) prevents the passage of therapeutics to their site of action. Polymeric drug delivery systems are promising solutions to effectively transport drugs into the brain. We recently showed that amphiphilic random copolymers based on the hydrophilic p(N-(2-hydroxypropyl)-methacrylamide), pHPMA, possessing randomly distributed hydrophobic p(laurylmethacrylate), pLMA, are able to mediate delivery of domperidone into the brain of mice in vivo. To gain further insight into structure-property relations, a library of carefully designed polymers based on p(HPMA) and p(LMA) was synthesized and tested applying an in vitro BBB model which consisted of human brain microvascular endothelial cells (HBMEC). Our model drug Rhodamine 123 (Rh123) exhibits, like domperidone, a low brain permeability since both substances are recognized by efflux transporters at the BBB. Transport studies investigating the impact of the polymer architecture in relation to the content of hydrophobic LMA revealed that random p(HPMA)-co-p(LMA) having 10mol% LMA is the most auspicious system. The copolymer significantly increased the permeability of Rh123 across the HBMEC monolayer whereas transcytosis of the polymer was very low. Further investigations on the mechanism of transport showed that integrity and barrier function of the BBB model were not harmed by the polymer. According to our results, p(HPMA)-co-p(LMA) copolymers are a promising delivery system for neurological therapeutics and their application might open alternative treatment strategies.


Subject(s)
Blood-Brain Barrier/metabolism , Drug Carriers/administration & dosage , Fluorescent Dyes/administration & dosage , Methacrylates/chemistry , Rhodamine 123/administration & dosage , Biological Transport/drug effects , Cell Line , Drug Carriers/chemistry , Humans , Models, Biological , Permeability/drug effects
4.
PLoS One ; 7(7): e41823, 2012.
Article in English | MEDLINE | ID: mdl-22860017

ABSTRACT

The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cß, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cß, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca(2+) signaling, PKCα/PKCßI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of soluble RAGE in the mouse lung. Our findings suggest that pharmacological stimulation of RAGE shedding might open alternative treatment strategies for Alzheimers disease and diabetes-induced inflammation.


Subject(s)
Receptors, Immunologic/metabolism , Receptors, Oxytocin/metabolism , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Receptors, Vasopressin/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM10 Protein , ADAM17 Protein , Adenylyl Cyclases/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Calcium Signaling , Cyclic AMP-Dependent Protein Kinases/metabolism , Dipeptides/pharmacology , Gene Knockdown Techniques , HEK293 Cells , Humans , Hydroxamic Acids/pharmacology , Lung/metabolism , MAP Kinase Signaling System , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/physiology , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide/physiology , Protease Inhibitors/pharmacology , Proteolysis , RNA Interference , Receptor for Advanced Glycation End Products
5.
J Biol Chem ; 283(51): 35507-16, 2008 Dec 19.
Article in English | MEDLINE | ID: mdl-18952609

ABSTRACT

The receptor for advanced glycation end products (RAGE) is a 55-kDa type I membrane glycoprotein of the immunoglobulin superfamily. Ligand-induced up-regulation of RAGE is involved in various pathophysiological processes, including late diabetic complications and Alzheimer disease. Application of recombinant soluble RAGE has been shown to block RAGE-mediated pathophysiological conditions. After expression of full-length RAGE in HEK cells we identified a 48-kDa soluble RAGE form (sRAGE) in the culture medium. This variant of RAGE is smaller than a 51-kDa soluble version derived from alternative splicing. The release of sRAGE can be induced by the phorbol ester PMA and the calcium ionophore calcimycin via calcium-dependent protein kinase C subtypes. Hydroxamic acid-based metalloproteinase inhibitors block the release of sRAGE, and by RNA interference experiments we identified ADAM10 and MMP9 to be involved in RAGE shedding. In protein biotinylation experiments we show that membrane-anchored full-length RAGE is the precursor of sRAGE and that sRAGE is efficiently released from the cell surface. We identified cleavage of RAGE to occur close to the cell membrane. Ectodomain shedding of RAGE simultaneously generates sRAGE and a membrane-anchored C-terminal RAGE fragment (RAGE-CTF). The amount of RAGE-CTF increases when RAGE-expressing cells are treated with a gamma-secretase inhibitor, suggesting that RAGE-CTF is normally further processed by gamma-secretase. Identification of these novel mechanisms involved in regulating the availability of cell surface-located RAGE and its soluble ectodomain may influence further research in RAGE-mediated processes in cell biology and pathophysiology.


Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Matrix Metalloproteinase 9/metabolism , Membrane Proteins/metabolism , Receptors, Immunologic/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , ADAM10 Protein , Alternative Splicing/drug effects , Alternative Splicing/genetics , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/genetics , Calcimycin/pharmacology , Carcinogens/pharmacology , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Diabetes Complications/genetics , Diabetes Complications/metabolism , Humans , Hydroxamic Acids/pharmacology , Ionophores/pharmacology , Matrix Metalloproteinase Inhibitors , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Protease Inhibitors/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary/genetics , RNA, Small Interfering/genetics , Receptor for Advanced Glycation End Products , Receptors, Immunologic/genetics , Tetradecanoylphorbol Acetate/pharmacology
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