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1.
Am J Hum Genet ; 96(3): 425-31, 2015 Mar 05.
Article in English | MEDLINE | ID: mdl-25683117

ABSTRACT

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.


Subject(s)
Craniosynostoses/genetics , Eye Abnormalities/genetics , Heterozygote , Hydrocephalus/genetics , Mutation, Missense , Osteogenesis Imperfecta/genetics , Procollagen-Proline Dioxygenase/genetics , Protein Disulfide-Isomerases/genetics , Child, Preschool , Female , Gene Frequency , Humans , Infant , Male , Pedigree , Procollagen-Proline Dioxygenase/metabolism , Protein Conformation , Protein Disulfide-Isomerases/metabolism , Protein Folding , Sequence Analysis, DNA
2.
Am J Hum Genet ; 96(6): 979-85, 2015 Jun 04.
Article in English | MEDLINE | ID: mdl-26027498

ABSTRACT

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.


Subject(s)
Models, Molecular , Mutation, Missense/genetics , Osteogenesis Imperfecta/genetics , Osteogenesis Imperfecta/pathology , Osteonectin/genetics , Amino Acid Sequence , Base Sequence , Collagen Type I/metabolism , Electrophoresis, Polyacrylamide Gel , Exome/genetics , Female , Genes, Recessive/genetics , Humans , Immunoblotting , Molecular Sequence Data , Mutagenesis, Site-Directed , Osteonectin/chemistry , Osteonectin/metabolism , Pedigree , Protein Conformation , Sequence Alignment , Sequence Analysis, DNA
3.
BMC Musculoskelet Disord ; 17: 89, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26891838

ABSTRACT

BACKGROUND: Aggrecan degradation is the hallmark of cartilage degeneration in osteoarthritis (OA), though it is unclear whether a common proteolytic process occurs in all individuals. METHODS: Aggrecan degradation in articular cartilage from the knees of 33 individuals with OA, who were undergoing joint replacement surgery, was studied by immunoblotting of tissue extracts. RESULTS: Matrix metalloproteinases (MMPs) and aggrecanases are the major proteases involved in aggrecan degradation within the cartilage, though the proportion of aggrecan cleavage attributable to MMPs or aggrecanases was variable between individuals. However, aggrecanases were more associated with the increase in aggrecan loss associated with OA than MMPs. While the extent of aggrecan cleavage was highly variable between individuals, it was greatest in areas of cartilage adjacent to sites of cartilage erosion compared to sites more remote within the same joint. Analysis of link protein shows that in some individuals additional proteolytic mechanisms must also be involved to some extent. CONCLUSIONS: The present studies indicate that there is no one protease, or a fixed combination of proteases, responsible for cartilage degradation in OA. Thus, rather than targeting the individual proteases for OA therapy, directing research to techniques that control global protease generation may be more productive.


Subject(s)
Aggrecans/analysis , Cartilage, Articular/chemistry , Osteoarthritis, Knee/diagnosis , Aged , Aged, 80 and over , Aggrecans/metabolism , Cartilage, Articular/metabolism , Female , Humans , Infant, Newborn , Male , Middle Aged , Osteoarthritis, Knee/metabolism
4.
Protein Expr Purif ; 91(1): 37-41, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23845403

ABSTRACT

The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.


Subject(s)
Cathepsin K/metabolism , Recombinant Proteins/metabolism , Amino Acid Sequence , Cathepsin K/chemistry , Cathepsin K/genetics , Collagen Type II/chemistry , Collagen Type II/metabolism , Humans , Methanol/chemistry , Molecular Sequence Data , Pichia/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
5.
Arthritis Rheum ; 64(5): 1477-87, 2012 May.
Article in English | MEDLINE | ID: mdl-22127564

ABSTRACT

OBJECTIVE: S100A8 and S100A9 are two Ca(2+) binding proteins classified as damage-associated molecular patterns or alarmins that are found in high amounts in the synovial fluid of osteoarthritis (OA) patients. The purpose of this study was to investigate whether S100A8 and/or S100A9 can interact with chondrocytes from OA patients to increase catabolic mediators. METHODS: Using immunohistochemistry, we stained for S100A8 and S100A9 protein, matrix metalloproteinases (MMPs), and a cartilage-breakdown epitope specific for MMPs (VDIPEN) in cartilage from OA donors. Isolated chondrocytes or explants from OA and non-OA donors were stimulated with S100A8 and/or S100A9. Messenger RNA and protein levels of MMPs, cytokines, and cartilage matrix molecules were determined with quantitative reverse transcription-polymerase chain reaction and Luminex techniques, respectively. For receptor blocking studies, specific inhibitors for Toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and carboxylated glycans were used. RESULTS: In cartilage from OA patients, the expression of S100A8 and S100A9 protein close to chondrocytes was associated with proteoglycan depletion and expression of MMP-1, MMP-3, and VDIPEN. Stimulation of chondrocytes with S100A8 and S100A9 caused a strong up-regulation of catabolic markers (MMPs 1, 3, 9, and 13, interleukin-6 [IL-6], IL-8, and monocyte chemotactic protein 1) and down-regulation of anabolic markers (aggrecan and type II collagen), thereby favoring cartilage breakdown. Blocking TLR-4, but not carboxylated glycans or RAGE, inhibited the S100 effect. The catabolic S100 effect was significantly more pronounced in chondrocytes from OA patients as compared to those from non-OA patients, possibly due to higher TLR-4 expression. CONCLUSION: S100A8 and S100A9 have a catabolic effect on human chondrocytes that is TLR-4 dependent. OA chondrocytes are more sensitive than normal chondrocytes to S100 stimulation.


Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Osteoarthritis/metabolism , Toll-Like Receptor 4/metabolism , Biomarkers/metabolism , Calgranulin A/administration & dosage , Calgranulin B/pharmacology , Cartilage Oligomeric Matrix Protein , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/pathology , Cytokines/genetics , Cytokines/metabolism , Epitopes/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Matrilin Proteins , Matrix Metalloproteinases/genetics , Matrix Metalloproteinases/metabolism , Oligopeptides/metabolism , Osteoarthritis/pathology , Peptide Fragments/metabolism , Receptor for Advanced Glycation End Products , Receptors, Immunologic/antagonists & inhibitors , Recombinant Proteins , Toll-Like Receptor 4/antagonists & inhibitors
6.
Dev Dyn ; 240(7): 1806-14, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21584905

ABSTRACT

Adamts4 encodes a widely-expressed proteinase thought to be involved in processes ranging from cartilage metabolism to ovarian follicle development. To study its physiological roles, Adamts4-null mice were created by gene targeting. Unexpectedly, these were found to be phenotypically normal, suggesting that other gene(s) may compensate for its loss. Adamts4(-/-) mice were, therefore, crossed with a strain lacking Adamts1, whose pattern of expression and substrate specificity overlap that of Adamts4. Most (>95%) Adamts1(-/-) ;Adamts4(-/-) mice died within 72 hr after birth with a marked thinning of the renal medulla. The renal defect was not observed in embryonic Adamts1(-/-) ;Adamts4(-/-) kidneys, but became apparent around birth. The few (<5%) Adamts1(-/-) ;Adamts4(-/-) animals to reach adulthood had the same renal phenotype seen in newborns. This study is thus the first to report Adamts4 expression and function in the mammalian kidney, and to demonstrate that Adamts1 and Adamts4 play redundant and essential roles in perinatal kidney development.


Subject(s)
ADAM Proteins/metabolism , Kidney Medulla/embryology , Kidney Medulla/metabolism , Procollagen N-Endopeptidase/metabolism , ADAM Proteins/genetics , ADAMTS1 Protein , ADAMTS4 Protein , Animals , Blotting, Western , Female , Immunohistochemistry , Male , Mice , Mice, Knockout , Procollagen N-Endopeptidase/genetics , Reverse Transcriptase Polymerase Chain Reaction
7.
J Immunol ; 182(8): 5024-31, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19342682

ABSTRACT

Multiple inflammatory mediators in osteoarthritis (OA) cartilage, including S100/calgranulin ligands of receptor for advanced glycation end products (RAGE), promote chondrocyte hypertrophy, a differentiation state associated with matrix catabolism. In this study, we observed that RAGE knockout was not chondroprotective in instability-induced knee OA in 8-wk-old mice. Hence, we tested the hypothesis that expression of the alternative S100/calgranulin and patterning receptor CD36, identified here as a marker of growth plate chondrocyte hypertrophy, mediates chondrocyte inflammatory and differentiation responses that promote OA. In rat knee joint destabilization-induced OA, RAGE expression was initially sparse throughout cartilage but increased diffusely by 4 wk after surgery. In contrast, CD36 expression focally increased at sites of cartilage injury and colocalized with developing chondrocyte hypertrophy and aggrecan cleavage NITEGE neoepitope formation. However, CD36 transfection in normal human knee-immortalized chondrocytes (CH-8 cells) was associated with decreased capacity of S100A11 and TNF-alpha to induce chondrocyte hypertrophy and ADAMTS-4 and matrix metalloproteinase 13 expression. S100A11 lost the capacity to inhibit proteoglycans synthesis and gained the capacity to induce proteoglycan synthesis in CD36-transfected CH-8 cells. Moreover, S100A11 required the p38 MAPK pathway kinase MKK3 to induce NITEGE development in mouse articular cartilage explants. However, CH-8 cells transfected with CD36 demonstrated decreased S100A11-induced MKK3 and p38 phosphorylation. Therefore, RAGE and CD36 patterning receptor expression were linked with opposing effects on inflammatory, procatabolic responses to S100A11 and TNF-alpha in chondrocytes.


Subject(s)
CD36 Antigens/immunology , Chondrocytes/immunology , Animals , Biomarkers/metabolism , Cell Differentiation , Cell Line , Chondrocytes/metabolism , Disease Models, Animal , Humans , Hypertrophy/immunology , Hypertrophy/metabolism , Hypertrophy/pathology , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , MAP Kinase Kinase 3/metabolism , Male , Mice , Mice, Knockout , Osteoarthritis, Knee/genetics , Osteoarthritis, Knee/immunology , Osteoarthritis, Knee/metabolism , Osteoarthritis, Knee/pathology , Rats , Receptor for Advanced Glycation End Products , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , S100 Proteins/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
8.
Lab Invest ; 90(1): 20-30, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19823173

ABSTRACT

Identification and characterization of therapeutic targets for joint conditions, such as osteoarthritis (OA), is exceedingly important for addressing the increasing burden of disease. Transforming growth factor-alpha (TGFalpha) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFalpha, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFalpha, rat chondrocytes and osteochondral explants were treated with TGFalpha and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFalpha resulted in the activation of RhoA/ROCK (Rho kinase), MEK (MAPK/ERK kinase)/ERK (extracellular-signal-regulated kinase), PI3K (phosphoinositide 3-kinase) and p38 MAPK (mitogen-activated protein kinase) pathways. Modification of the chondrocyte actin cytoskeleton was stimulated by TGFalpha, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFalpha suppressed expression of anabolic genes including Sox9, type II collagen and aggrecan, which were rescued only by inhibiting MEK/ERK activation. Furthermore, catabolic factor upregulation by TGFalpha was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-alpha, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFalpha to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFalpha. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFalpha. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFalpha. These data suggest that TGFalpha is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.


Subject(s)
Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Mitogen-Activated Protein Kinase Kinases/metabolism , Transforming Growth Factor alpha/pharmacology , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Aggrecans/metabolism , Animals , Bone and Bones/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/metabolism , Collagen Type II/metabolism , Enzyme Activation , Extracellular Matrix/drug effects , Extracellular Matrix/genetics , Gene Expression Regulation , Humans , Intracellular Membranes/metabolism , Male , Metabolism/genetics , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , rho-Associated Kinases/antagonists & inhibitors , rhoA GTP-Binding Protein/antagonists & inhibitors
9.
Hum Mol Genet ; 17(13): 1904-15, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18344557

ABSTRACT

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.


Subject(s)
Hyaluronoglucosaminidase/genetics , Hyaluronoglucosaminidase/metabolism , Mucopolysaccharidoses/physiopathology , Osteoarthritis/physiopathology , Animals , Disease Models, Animal , Female , Gene Targeting , Glycosaminoglycans/metabolism , Humans , Hyaluronic Acid/blood , Joints/pathology , Male , Mice , Mice, Knockout , Mucopolysaccharidoses/complications , Mucopolysaccharidoses/genetics , Osteoarthritis/complications , Osteoarthritis/genetics , Osteoarthritis/metabolism , Phenotype
10.
Am J Pathol ; 173(1): 161-9, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18511517

ABSTRACT

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.


Subject(s)
Cartilage, Articular/metabolism , Cathepsins/metabolism , Collagen Type II/metabolism , Osteoarthritis, Knee/metabolism , Adolescent , Adult , Aged , Aging/physiology , Base Sequence , Biphenyl Compounds/pharmacology , Cartilage, Articular/physiopathology , Cathepsin K , Cathepsins/drug effects , Collagen Type II/drug effects , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Humans , Middle Aged , Osteoarthritis, Knee/physiopathology , Peptides/drug effects , Peptides/metabolism
11.
Matrix Biol ; 27(8): 653-60, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18762256

ABSTRACT

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.


Subject(s)
Hyaluronic Acid/metabolism , Hyaluronoglucosaminidase/deficiency , Hyaluronoglucosaminidase/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Animals , Gene Deletion , Gene Expression Regulation, Enzymologic , Hyaluronoglucosaminidase/genetics , Mice , Mice, Knockout , Phenotype , Transcription, Genetic/genetics
12.
J Mol Biol ; 371(1): 137-53, 2007 Aug 03.
Article in English | MEDLINE | ID: mdl-17561110

ABSTRACT

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.


Subject(s)
Cathepsins/antagonists & inhibitors , Cathepsins/chemistry , Cysteine Endopeptidases/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cathepsin L , Cathepsins/genetics , Cathepsins/metabolism , Crystallography, X-Ray , Cystatins/chemistry , Cystatins/genetics , Cystatins/metabolism , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
13.
Mol Biol Cell ; 16(11): 5215-26, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16135528

ABSTRACT

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.


Subject(s)
Membrane Proteins/physiology , Proprotein Convertase 5/metabolism , Serine Endopeptidases/metabolism , Tissue Inhibitor of Metalloproteinases/metabolism , Animals , CHO Cells , COS Cells , Cell Line , Chlorocebus aethiops , Cricetinae , Cricetulus , Cysteine , Heparitin Sulfate/physiology , Humans , Membrane Proteins/metabolism , Metalloproteases/metabolism , Mice , Proprotein Convertase 5/physiology , Proprotein Convertases , Protein Processing, Post-Translational , Protein Structure, Tertiary/physiology , Serine Endopeptidases/physiology , Transfection
14.
Bone ; 117: 23-30, 2018 12.
Article in English | MEDLINE | ID: mdl-30217615

ABSTRACT

The major organic component of bone is collagen type I. Osteoclasts are terminally differentiated multinucleated cells of hematopoietic origin that are essential for physiological development of bone and teeth. We examined if osteoclast differentiation from murine bone marrow precursors is affected by collagen type I, or by its degradation products produced by human recombinant cathepsin K. Osteoclasts formation was dose-dependently inhibited in the presence of full length collagen type I or its 30-75 kDa degradation products added to the osteoclast differentiation media for the duration of an experiment. Collagen degradation fragments signaled through SH-2 phosphatases, inhibiting calcium signaling and NFATc1 translocation in osteoclast precursors. Osteoclasts and their precursors expressed a collagen receptor of leukocyte receptor complex family, LAIR-1. Importantly, collagen fragments failed to inhibit osteoclast formation from LAIR-1 deficient murine osteoclast precursors. This study demonstrates that collagen degradation fragments inhibit osteoclast formation acting through LAIR-1, providing a novel mechanism for the physiologically-relevant negative control of osteoclastogenesis.


Subject(s)
Collagen Type I/metabolism , Feedback, Physiological , Osteoclasts/metabolism , Receptors, Immunologic/metabolism , Animals , Bone Marrow Cells/metabolism , Humans , Male , Mice , Osteogenesis , Peptide Fragments/metabolism , Signal Transduction
15.
Methods Mol Med ; 135: 201-9, 2007.
Article in English | MEDLINE | ID: mdl-17951660

ABSTRACT

Quantitation of glycosaminoglycans (GAGs) in the form of aggrecan fragments released from cartilage in culture is a simple way to determine the efficacy of different cytokines alone or in combination in simulating cartilage catabolism. Two approaches for GAG assay are described, with special attention being paid to the advantages and limitations of each method.


Subject(s)
Cartilage/metabolism , Glycosaminoglycans/metabolism , Aggrecans/metabolism , Animals , Carbazoles/analysis , Cartilage/drug effects , Cattle , Coloring Agents , Cytokines/pharmacology , Glycosaminoglycans/analysis , Glycosaminoglycans/standards , Methylene Blue/analogs & derivatives , Reference Standards , Tissue Culture Techniques , Uronic Acids/analysis
16.
J Histochem Cytochem ; 54(9): 965-80, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16709729

ABSTRACT

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.


Subject(s)
Bone Development , Cartilage/enzymology , Matrix Metalloproteinase 9/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Cartilage/growth & development , Cell Line, Tumor , Cysteine/metabolism , Enzyme Activation , Epiphyses/enzymology , Epiphyses/growth & development , Epitopes , Humans , Hydrolysis , Immunohistochemistry , Mice , Microscopy, Electron , Molecular Sequence Data , Peptides/metabolism , Protein Precursors/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism
17.
Matrix Biol ; 24(5): 371-5, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15970436

ABSTRACT

Culture medium obtained from baculovirus-infected High Five insect cells contains an endoglycosidase activity capable of releasing chondroitin sulfate chains from aggrecan, decorin and biglycan. Release appears to occur by cleavage within the linkage region of the chondroitin sulfate chain, but not all chains are amenable to release. Culture medium from Sf9 insect cells does not contain this activity. The endoglycosidase may become a useful reagent for biochemical research for releasing intact chondroitin sulfate chains from proteoglycans. It may also represent an impediment to such research when baculovirus systems are used to generate recombinant proteoglycans.


Subject(s)
Chondroitin Sulfate Proteoglycans/chemistry , Chondroitin Sulfate Proteoglycans/metabolism , Chondroitin Sulfates/metabolism , Culture Media, Conditioned/chemistry , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Glycoside Hydrolases/metabolism , Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Spodoptera/cytology , Aggrecans , Animals , Cattle
18.
Biochem J ; 375(Pt 1): 183-9, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-12859252

ABSTRACT

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.


Subject(s)
Extracellular Matrix Proteins , Metalloendopeptidases/metabolism , Proteoglycans/chemistry , Proteoglycans/metabolism , ADAM Proteins , ADAMTS4 Protein , ADAMTS5 Protein , Aged , Aggrecans , Animals , Cartilage/chemistry , Cartilage/embryology , Cartilage/growth & development , Cattle , Humans , Infant, Newborn , Lectins, C-Type , Procollagen N-Endopeptidase , Protein Structure, Tertiary
19.
Arthritis Rheumatol ; 67(10): 2691-701, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26138996

ABSTRACT

OBJECTIVE: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA. METHODS: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C). RESULTS: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage. CONCLUSION: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.


Subject(s)
Chemokine CCL2/antagonists & inhibitors , Disease Progression , Osteoarthritis/prevention & control , Osteoarthritis/physiopathology , Signal Transduction/physiology , Wounds and Injuries/complications , Animals , Benzoxazines/pharmacology , Cartilage, Articular/pathology , Cartilage, Articular/physiopathology , Chemokine CCL2/drug effects , Chemokine CCL2/physiology , Disease Models, Animal , Male , Osteoarthritis/etiology , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Spiro Compounds/pharmacology , Transforming Growth Factor alpha/antagonists & inhibitors , Transforming Growth Factor alpha/drug effects , Transforming Growth Factor alpha/physiology , Tyrphostins/pharmacology , Up-Regulation/physiology
20.
Arthritis Rheumatol ; 67(8): 2164-75, 2015 May.
Article in English | MEDLINE | ID: mdl-25891852

ABSTRACT

OBJECTIVE: High-frequency, low-amplitude whole-body vibration (WBV) is being used to treat a range of musculoskeletal disorders; however, there is surprisingly limited knowledge regarding its effect(s) on joint tissues. This study was undertaken to examine the effects of repeated exposure to WBV on bone and joint tissues in an in vivo mouse model. METHODS: Ten-week-old male mice were exposed to vertical sinusoidal vibration under conditions that mimic those used clinically in humans (30 minutes per day, 5 days per week, at 45 Hz with peak acceleration at 0.3g). Following WBV, skeletal tissues were examined by micro-computed tomography, histologic analysis, and immunohistochemistry, and gene expression was quantified using real-time polymerase chain reaction. RESULTS: Following 4 weeks of WBV, intervertebral discs showed histologic hallmarks of degeneration in the annulus fibrosus, disruption of collagen organization, and increased cell death. Greater Mmp3 expression in the intervertebral disc, accompanied by enhanced collagen and aggrecan degradation, was found in mice exposed to WBV as compared to controls. Examination of the knee joints after 4 weeks of WBV revealed meniscal tears and focal damage to the articular cartilage, changes resembling osteoarthritis. Moreover, mice exposed to WBV also demonstrated greater Mmp13 gene expression and enhanced matrix metalloproteinase-mediated collagen and aggrecan degradation in articular cartilage as compared to controls. No changes in trabecular bone microarchitecture or density were detected in the proximal tibia. CONCLUSION: Our experiments reveal significant negative effects of WBV on joint tissues in a mouse model. These findings suggest the need for future studies of the effects of WBV on joint health in humans.


Subject(s)
Cartilage, Articular/injuries , Intervertebral Disc/injuries , Leg Injuries/etiology , RNA, Messenger/metabolism , Spinal Injuries/etiology , Tibia/injuries , Tibial Meniscus Injuries , Vibration/adverse effects , Aggrecans/metabolism , Animals , Bone Density , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Collagen/metabolism , Gene Expression Profiling , Intervertebral Disc/metabolism , Intervertebral Disc/pathology , Leg Injuries/diagnosis , Leg Injuries/metabolism , Male , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3/metabolism , Menisci, Tibial/metabolism , Menisci, Tibial/pathology , Mice , Radiography , Reverse Transcriptase Polymerase Chain Reaction , Spinal Injuries/diagnosis , Spinal Injuries/metabolism , Tibia/diagnostic imaging
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