RESUMO
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 30-75â¯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.
Assuntos
Colágeno Tipo I/metabolismo , Retroalimentação Fisiológica , Osteoclastos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Células da Medula Óssea/metabolismo , Humanos , Masculino , Camundongos , Osteogênese , Fragmentos de Peptídeos/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Agrecanas/análise , Cartilagem Articular/química , Osteoartrite do Joelho/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/metabolismoRESUMO
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.
Assuntos
Modelos Moleculares , Mutação de Sentido Incorreto/genética , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Osteonectina/genética , Sequência de Aminoácidos , Sequência de Bases , Colágeno Tipo I/metabolismo , Eletroforese em Gel de Poliacrilamida , Exoma/genética , Feminino , Genes Recessivos/genética , Humanos , Immunoblotting , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Osteonectina/química , Osteonectina/metabolismo , Linhagem , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
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.
Assuntos
Catepsina K/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Catepsina K/química , Catepsina K/genética , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Humanos , Metanol/química , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually due to dominant mutations in COL1A1 or COL1A2. Rare recessive forms of OI, caused by mutations in genes involved in various aspects of bone formation, have been described as well. OBJECTIVE: To identify the cause of OI in eight children with severe bone fragility and a clinical diagnosis of OI type IV who had had negative results on COL1A1/COL1A2 Sanger sequencing. METHODS: Whole exome sequencing was performed in genomic DNA samples from all eight individuals. RESULTS: WNT1 mutations were found in four children from three families. WNT1 was the only gene where mutations were found in all of these four patients. Two siblings from a consanguineous family had a homozygous missense mutation affecting a highly conserved cysteine residue in WNT1 (c.428G>T (p.Cys143Phe)). One girl had a homozygous frameshift deletion (c.287_300del(p.Gln96Profs)). A girl from a third family was compound heterozygous for a frameshift insertion and a missense mutation affecting a conserved amino acid (c.946_949insAACA (p.Ser317Lysfs); c.1063G>T (p.Val355Phe)). All of these children had short stature, low bone density, and severe vertebral compression fractures in addition to multiple long bone fractures in the first years of life. The Wnt signalling pathway is one of the key regulators of osteoblast activity. CONCLUSIONS: Recessive inactivating mutations in WNT1 are a new cause of OI type IV.
Assuntos
Osso e Ossos/diagnóstico por imagem , Modelos Moleculares , Osteogênese Imperfeita/genética , Conformação Proteica , Proteína Wnt1/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Mutação da Fase de Leitura/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Radiografia , Análise de Sequência de DNA , Proteína Wnt1/químicaRESUMO
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.
Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/metabolismo , Receptor 4 Toll-Like/metabolismo , Biomarcadores/metabolismo , Calgranulina A/administração & dosagem , Calgranulina B/farmacologia , Proteína de Matriz Oligomérica de Cartilagem , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Citocinas/genética , Citocinas/metabolismo , Epitopos/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Proteínas Matrilinas , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Oligopeptídeos/metabolismo , Osteoartrite/patologia , Fragmentos de Peptídeos/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/antagonistas & inibidores , Proteínas Recombinantes , Receptor 4 Toll-Like/antagonistas & inibidoresRESUMO
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.
Assuntos
Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador alfa/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Agrecanas/metabolismo , Animais , Osso e Ossos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Ativação Enzimática , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Regulação da Expressão Gênica , Humanos , Membranas Intracelulares/metabolismo , Masculino , Metabolismo/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
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.
Assuntos
Antígenos CD36/imunologia , Condrócitos/imunologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Condrócitos/metabolismo , Modelos Animais de Doenças , Humanos , Hipertrofia/imunologia , Hipertrofia/metabolismo , Hipertrofia/patologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , MAP Quinase Quinase 3/metabolismo , Masculino , Camundongos , Camundongos Knockout , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/imunologia , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas S100/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.
Assuntos
Catepsina B/antagonistas & inibidores , Cistatinas/química , Proteínas de Protozoários/química , Animais , Cistatinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Proteínas de Protozoários/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Soluções , Trypanosoma cruziRESUMO
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.
Assuntos
Cartilagem Articular/metabolismo , Catepsinas/metabolismo , Colágeno Tipo II/metabolismo , Osteoartrite do Joelho/metabolismo , Adolescente , Adulto , Idoso , Envelhecimento/fisiologia , Sequência de Bases , Compostos de Bifenilo/farmacologia , Cartilagem Articular/fisiopatologia , Catepsina K , Catepsinas/efeitos dos fármacos , Colágeno Tipo II/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Pessoa de Meia-Idade , Osteoartrite do Joelho/fisiopatologia , Peptídeos/efeitos dos fármacos , Peptídeos/metabolismoRESUMO
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.
Assuntos
Catepsinas/antagonistas & inibidores , Catepsinas/química , Cisteína Endopeptidases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catepsina L , Catepsinas/genética , Catepsinas/metabolismo , Cristalografia por Raios X , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Desenvolvimento Ósseo , Cartilagem/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Cartilagem/crescimento & desenvolvimento , Linhagem Celular Tumoral , Cisteína/metabolismo , Ativação Enzimática , Epífises/enzimologia , Epífises/crescimento & desenvolvimento , Epitopos , Humanos , Hidrólise , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Dados de Sequência Molecular , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Proteínas de Membrana/fisiologia , Pró-Proteína Convertase 5/metabolismo , Serina Endopeptidases/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Cisteína , Heparitina Sulfato/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Camundongos , Pró-Proteína Convertase 5/fisiologia , Pró-Proteína Convertases , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína/fisiologia , Serina Endopeptidases/fisiologia , TransfecçãoRESUMO
Increased expression of metalloproteinases is a fundamental aspect of arthritispathology and its control is a major therapeutic objective. In cartilage cultured in the presence of the cytokines interleukin-1 and oncostatin M, chondrocytes produce enhanced levels of metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and MMP (matrix metalloproteinase) families, resulting in the degradation of aggrecan and collagen. The histone deacetylase inhibitors trichostatin A and butyrate were shown to drastically reduce expression of these enzymes relatively selectively, with concomitant inhibition of breakdown of matrix components. This family of enzymes is therefore a promising target for therapeutic intervention.
Assuntos
Doenças das Cartilagens/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/administração & dosagem , Inibidores de Histona Desacetilases , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/enzimologia , Doenças das Cartilagens/enzimologia , Histona Desacetilases/metabolismo , HumanosRESUMO
Tissue inhibitor of metalloproteinases-3 (TIMP-3) is known to inhibit matrix metalloproteinases, aggrecanases, and tumor necrosis factor (TNF)-alpha-converting enzyme (TACE, ADAM17). These metalloproteases participate in different aspects of joint destruction in inflammatory arthritis. To determine the relative importance of this inhibitor in joint pathology, wild-type and Timp3-/- mice were immunized with methylated bovine serum albumin followed by arthritis induction by intra-articular injection of the same antigen. Animals were monitored for up to 14 days after challenge, and joint tissues were analyzed by routine and Safranin O staining and for the presence of aggrecan neoepitopes produced by metalloprotease cleavage. Serum TNF-alpha was measured by immunoassay. Compared to wild-type animals, Timp3-/- mice showed a dramatic increase in the initial inflammatory response to intra-articular antigen injection, and serum TNF-alpha levels were greatly elevated in the Timp3-/- animals after immunization. However, these differences in clinical features disappeared by days 7 to 14. No difference in Safranin O staining or aggrecan cleavage site neoepitope abundance was seen. Thus, in inflammatory joint disease TIMP-3 likely dampens the inflammatory response of TNF-alpha by reducing ADAM17 activity.
Assuntos
Artrite/patologia , Inflamação/patologia , Inibidor Tecidual de Metaloproteinase-3/deficiência , Fator de Necrose Tumoral alfa/análise , Proteínas ADAM , Proteína ADAM17 , Agrecanas , Animais , Antígenos/administração & dosagem , Artrite/induzido quimicamente , Corantes , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Imuno-Histoquímica , Inflamação/induzido quimicamente , Injeções Intra-Articulares , Lectinas Tipo C , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Knockout , Fenazinas , Proteoglicanas/metabolismo , Soroalbumina Bovina/administração & dosagemRESUMO
The use of synthetic peptides to generate rabbit polyclonal anticatabolic neoepitope antibodies that can be used to study the presence of defined proteolytic cleavage sites in aggrecan is described. Principles of peptide design and methods for preparation and characterization of ovalbumin conjugates are presented along with approaches for the characterization and affinity purification of the resulting antisera. Limitations associated with the use of antipeptide antibodies to study authentic protein neoepitopes are discussed.
Assuntos
Epitopos/imunologia , Proteínas da Matriz Extracelular/imunologia , Soros Imunes/biossíntese , Peptídeos/imunologia , Proteoglicanas/imunologia , Agrecanas , Sequência de Aminoácidos , Animais , Anticorpos/análise , Anticorpos/imunologia , Epitopos/química , Proteínas da Matriz Extracelular/química , Soros Imunes/isolamento & purificação , Lectinas Tipo C , Dados de Sequência Molecular , Peptídeos/química , Proteoglicanas/química , Coelhos , Succinimidas/químicaRESUMO
The cathepsins are a family of cysteine proteases that have been broadly implicated in proteolytic processes during cell growth, cell development, and normal adult cellular function. Cathepsin L is a major secretory product of rat and mouse Sertoli cells, the absence of which in furless mice is associated with atrophy of some seminiferous tubules. However, furless mice produce viable sperm, suggesting the possibility that other members of the cathepsin family of proteases may complement cathepsin L action in the testis. Our objective herein was to begin to test this hypothesis. To this end, we first utilized cDNA microarray technology to identify the members of the cathepsin gene family expressed by freshly isolated adult rat Sertoli cells. This approach, complemented by Northern blot analyses, showed that in addition to cathepsin L, cathepsin K is highly and specifically expressed in Sertoli cells. As is also true of cathepsin L, cathepsin K mRNA was found to be expressed by Sertoli cells at specific stages of the cycle of the seminiferous epithelium, with maximal expression at stages VI-VII. The use of immunocytochemical methods revealed that cathepsin K protein localizes to the cytoplasm of Sertoli cells at stages VI-VIII, to small punctuate lysosomes at stages I-VIII and XIII-XIV, and to early and late residual bodies at stages IX-XII. This localization was found to be similar to that of cathepsin L. The similarity in the expression and localization of cathepsin K and cathepsin L suggest that the two proteases may have similar functions. If true, this might explain the fertility of furless mice. Further, the results suggest that cathepsin K, in both its secreted and lysosomal forms, may play a role in the degradation of Sertoli cell residual bodies.
Assuntos
Catepsinas/genética , Catepsinas/metabolismo , Células de Sertoli/enzimologia , Ácidos/metabolismo , Fatores Etários , Animais , Catepsina K , Catepsina L , Cisteína Endopeptidases , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Túbulos Seminíferos/enzimologia , Ativação TranscricionalRESUMO
Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.
Assuntos
Catequina/análogos & derivados , Catequina/metabolismo , Desintegrinas/antagonistas & inibidores , Ésteres/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Proteínas ADAM , Proteína ADAM10 , Proteína ADAMTS1 , Proteína ADAMTS4 , Proteína ADAMTS5 , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Western Blotting , Bovinos , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Desintegrinas/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Concentração Inibidora 50 , Insetos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Pró-Colágeno N-Endopeptidase , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , CháRESUMO
OBJECTIVE: To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor alpha-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice. METHODS: Transgenic mice constitutively expressing TSG-6 protein in cartilage were generated. Cartilage-specific constitutive expression of TSG-6 protein was confirmed by in situ hybridization, Western blot analysis, and immunohistochemistry. Control and transgenic mice were immunized with methylated bovine serum albumin (mBSA), and arthritis was induced by the intraarticular injection of mBSA. Mice were monitored up to day 35 after the challenge, and knee joint sections were examined for loss of cartilage proteoglycan (aggrecan) using Safranin O staining and antibodies to neoepitopes generated by various metalloproteinases (MPs). The loss of aggrecan in Safranin O-stained sections was quantified by morphometric methods. RESULTS: Tsg6/tnfip6 transgenic mice constitutively expressed tsg6/tnfip6 messenger RNA and corresponding TSG-6 protein in cartilage from embryonic life through adulthood, without any phenotypic abnormalities. These mice were used for AIA studies. Intraarticular injection of mBSA uniformly induced severe inflammation both in control (wild-type and an irrelevant transgenic line) mice and in tsg6/tnfip6 transgenic mice. In contrast to the mBSA-injected knee joints of control animals that were heavily damaged from day 5, the cartilage of transgenic mice that constitutively expressed TSG-6 protein remained intact for at least 1 week, and this was followed by a relatively reduced loss of aggrecan. Concomitant with the loss of aggrecan, MP-generated neoepitopes accumulated in unprotected joints. By day 35, the proteoglycan content returned to nearly normal levels in tsg6/tnfip6 transgenic mice, whereas it remained low in MP-damaged knee cartilage of control mice. CONCLUSION: TSG-6 protein is known to form a complex with inter-alpha-inhibitor (IalphaI), a potent serine protease inhibitor, which may be immobilized via the hyaluronan (HA)-binding domain of TSG-6 protein in the HA-rich extracellular matrix of cartilage. Thus, the local accumulation of TSG-6 protein and TSG-6 protein-bound IalphaI in tsg6/tnfip6 transgenic mice may inhibit serine proteases and subsequent activation of MPs. It is suggested that this mechanism might protect cartilage from extensive degradation even in the presence of acute inflammation.
Assuntos
Anti-Inflamatórios/metabolismo , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Cartilagem Articular/metabolismo , Moléculas de Adesão Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Artrite Experimental/patologia , Western Blotting , Cartilagem Articular/citologia , Moléculas de Adesão Celular/genética , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Clonagem Molecular , Humanos , Imuno-Histoquímica , Hibridização In Situ , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/genéticaRESUMO
The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.