Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.
ADAM Proteins ; antagonists & inhibitors ; biosynthesis ; genetics ; ADAM17 Protein ; Amyloid Precursor Protein Secretases ; Animals ; Humans ; Mice ; Peptide Library ; Peptides ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4).
ADAM Proteins ; antagonists & inhibitors ; chemistry ; genetics ; physiology ; Aggrecans ; metabolism ; Alternative Splicing ; Arthritis ; enzymology ; genetics ; Cartilage ; enzymology ; Endopeptidases ; genetics ; physiology ; Extracellular Matrix ; enzymology ; Humans ; Protein Structure, Tertiary

The effects of inhibitors of TNF alpha converting enzyme (TACE) on TNF alpha secretion were studied to develop an approach to interfere inflammation processes. The HL-60 cell lines were stimulated in vitro with LPS intravenously for different time to establish the cellular model of inflammation and simultaneously induce in vivo inflammation animal model by LPS The cytotoxic effects of soluble TNF alpha were checked using MTT colorimetric method to determine the rate of cell proliferation. The level of expression of TACE was detected by using RT-PCR, FCM and immuno-histochemical technique respectively. It was found Chinese medicine Reduqing (RDQ) could inhibit the transcription of TNF alpha mRNA induced by LPS stimulation (P < 0.01, compared with the control). The antioligodeoxyribonucleotide (anti-ODN) of TNF alpha mRNA could inhibit 78.9% of TNF alpha secretion. The mimic peptides of TACE substrates with hydroxamine group showed potency in vivo and in vitro against converting of pro-TNF alpha. It was concluded that all the three types of TACE inhibitors can regulate the expression of TACE at different levels and inhibit sTNF alpha secretion, indicating TACE is a novel target for inflammation therapy.
ADAM Proteins ; ADAM17 Protein ; Drugs, Chinese Herbal ; pharmacology ; HL-60 Cells ; Humans ; Inflammation ; metabolism ; Lipopolysaccharides ; Metalloendopeptidases ; antagonists & inhibitors ; Oligodeoxyribonucleotides ; antagonists & inhibitors ; Protein Precursors ; antagonists & inhibitors ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors ; genetics ; metabolism ; secretion

ADAM Proteins ; genetics ; ADAM17 Protein ; Adrenergic beta-Antagonists ; therapeutic use ; Animals ; Male ; Metoprolol ; therapeutic use ; Myocardial Infarction ; drug therapy ; physiopathology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; genetics ; Ventricular Function, Left ; drug effects ; Ventricular Remodeling ; drug effects

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors