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1.
Peptides ; 27(7): 1877-85, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16574278

ABSTRACT

Beta-secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel "bis-statine" inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.


Subject(s)
Amino Acids/chemistry , Biochemistry/methods , Endopeptidases/chemistry , Peptides/chemistry , Protease Inhibitors/pharmacology , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Animals , Biotinylation , CHO Cells , Cricetinae , Crystallization , Crystallography , Models, Molecular , Molecular Sequence Data , Protein Conformation
2.
Matrix Biol ; 23(4): 219-30, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15296936

ABSTRACT

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.


Subject(s)
Cartilage, Articular/enzymology , Endopeptidases/metabolism , Metalloendopeptidases/metabolism , ADAM Proteins , ADAMTS9 Protein , Aggrecans , Animals , Antibodies, Monoclonal/immunology , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , Enzyme-Linked Immunosorbent Assay/methods , Extracellular Matrix Proteins/metabolism , Humans , Lectins, C-Type , Metalloendopeptidases/genetics , Metalloendopeptidases/immunology , Metalloendopeptidases/isolation & purification , Osteoarthritis/metabolism , Polymerase Chain Reaction , Proteoglycans/metabolism , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Tissue Distribution
3.
Osteoarthritis Cartilage ; 12(8): 599-613, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15262240

ABSTRACT

INTRODUCTION: Articular cartilage matrix synthesis and degradation are dynamic processes that must be balanced for proper maintenance of the tissue. In osteoarthritis (OA), this balance is skewed toward degradation and ultimate loss of matrix. The transcriptional and/or activity levels of hundreds of genes are dysregulated in chondrocytes from osteoarthritic cartilage, and a subset of these genes may represent pivotal factors that could be modulated if their specific role in the disease process could be identified. OBJECTIVE: To investigate the role of ADAMTS-4 and ADAMTS-5 in cartilage matrix degradation by developing a chondrocyte pellet culture assay in combination with adenoviral gene expression, and to demonstrate the utility of this assay by assessing the specific functional contribution of these genes to cartilage matrix metabolism. METHODS: A full-length cDNA for bovine ADAMTS-4 (bADAMTS-4) was isolated, and used to evaluate the expression, regulation, and activity of this gene in bovine cartilage. Adenoviruses expressing bADAMTS-4, human ADAMTS-5 (hADAMTS-5) or human bone morphogenetic protein 2 (BMP-2) were used to infect primary chondrocytes, and their effect on extracellular matrix metabolism was assessed by monitoring the accumulation and release of glycosaminoglycans (GAG) in three-dimensional chondrocyte pellet cultures. RESULTS: Analysis of bADAMTS-4 transcriptional regulation in chondrocytes revealed that interleukin-1alpha (IL-1alpha) was the most potent inducer of bADAMTS-4 mRNA and subsequent aggrecan degradation in cartilage explant cultures of those cytokines tested. bADAMTS-4 mRNA induction by IL-1alpha was greater in nasal cartilage than in articular cartilage. Chondrocytes infected with adenovirus expressing either bADAMTS-4 or hADAMTS-5 genes showed increased aggrecan degradation in newly synthesized matrix by pellet cultures while chondrocytes overexpressing BMP-2 showed increased aggrecan synthesis. CONCLUSION: Adenoviral delivery of genes to primary bovine chondrocytes, followed by culture in three-dimensional pellet format and evaluation of extracellular matrix protein metabolism, is a useful functional assay for assessing the role of genes on cartilage matrix synthesis and degradation.


Subject(s)
Cartilage, Articular/enzymology , Chondrocytes/enzymology , Metalloendopeptidases/metabolism , ADAM Proteins , ADAMTS4 Protein , ADAMTS5 Protein , Adenoviridae/genetics , Amino Acid Sequence , Animals , Base Sequence , Cartilage, Articular/cytology , Cattle , Cells, Cultured , Gene Expression Regulation, Enzymologic , Gene Transfer Techniques , Genetic Vectors/genetics , Humans , Metalloendopeptidases/genetics , Molecular Sequence Data , Procollagen N-Endopeptidase , RNA, Messenger/genetics , Species Specificity
4.
Int Immunopharmacol ; 4(5): 693-708, 2004 May.
Article in English | MEDLINE | ID: mdl-15120653

ABSTRACT

Interleukin 22 (IL-22) is a cytokine induced during both innate and adaptive immune responses. It can effect an acute phase response, implicating a role for IL-22 in mechanisms of inflammation. IL-22 requires the presence of the IL-22 receptor (IL-22R) and IL-10 receptor 2 (IL-10R2) chains, two members of the class II cytokine receptor family (CRF2), to effect signal transduction within a cell. We studied the interaction between human IL-22 and the extracellular domains (ECD) of its receptor chains in an enzyme-linked immunoabsorbant assay (ELISA)-based format, using biotinylated IL-22 (bio-IL-22) and receptor-fusions containing the ECD of a receptor fused to the Fc of hIgG1 (IL-22R-Fc and IL-10R2-Fc). IL-22 has measurable affinity for IL-22R-Fc homodimer and undetectable affinity for IL-10R2. IL-22 has substantially greater affinity for IL-22R/IL-10R2-Fc heterodimers. Further analyses involving sequential additions of receptor homodimers and cytokine indicates that the IL-10R2(ECD) binds to a surface created by the interaction between IL-22 and the IL-22R(ECD), and thereby further stabilizes the association of IL-22 within this cytokine-receptor-Fc complex. Both a neutralizing rat monoclonal antibody, specific for human IL-22, and human IL-22BP-Fc, an Fc-fusion of the secreted IL-22 binding-protein and proposed natural antagonist for IL-22, bind to similar cytokine epitopes that may overlap the binding site for IL-22R(ECD). Another rat monoclonal antibody, specific for IL-22, binds to an epitope that may overlap a separate binding site for IL-10R2(ECD). We propose, based on this data, a temporal model for the development of a functional IL-22 cytokine-receptor complex.


Subject(s)
Interleukins/metabolism , Receptors, Interleukin/metabolism , Animals , CHO Cells , Cricetinae , Dimerization , Enzyme-Linked Immunosorbent Assay/methods , Humans , Interleukins/pharmacology , Receptors, Interleukin/drug effects , Receptors, Interleukin-10 , Time Factors , Interleukin-22
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