Publications (17) View all
-
Article: Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library.
[show abstract] [hide abstract]
ABSTRACT: The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.Journal of Biological Chemistry 11/2000; 275(40):31422-7. · 4.77 Impact Factor -
Article: Replication-associated activities of purified human papillomavirus type 11 E1 helicase.
[show abstract] [hide abstract]
ABSTRACT: Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.Protein Expression and Purification 04/2000; 18(2):148-59. · 1.59 Impact Factor -
Article: Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha.
[show abstract] [hide abstract]
ABSTRACT: Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. We have purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production.Nature 03/1997; 385(6618):733-6. · 36.28 Impact Factor -
Article: Structural and functional characterization of the HPV16 E7 protein expressed in bacteria.
[show abstract] [hide abstract]
ABSTRACT: The E7 gene of the human papillomaviruses (HPV) encodes a 98-amino acid, multifunctional nuclear phosphoprotein with functional and structural similarities to adenovirus E1A and the papovavirus T antigens. E7 is a viral oncoprotein, which will cooperate with an activated ras oncogene to transform primary rodent cells, and can cooperate with the HPV E6 protein for the efficient immortalization of primary human keratinocytes. Due to the compelling epidemiological and experimental association between HPV infection and cervical cancer, we have undertaken a detailed study of the structure of the HPV16 E7 protein. The E7 protein was expressed in Escherichia coli as a native, unfused polypeptide, and soluble protein was purified by conventional chromatographic techniques. The purified protein was assessed for various biochemical and biophysical properties. Purified E7 binds the retinoblastoma protein avidly and specifically, and it can dissociate the E2F transcription factor when assayed in vitro. Circular dichroism spectroscopy indicated that E7 reversibly binds Zn2+ and Cd2+, resulting in a substantial increase in the alpha-helical content of the metal-bound E7 consistent with the stabilization of a hydrophobic core in the COOH terminus of the protein.Journal of Biological Chemistry 01/1994; 268(34):26018-25. · 4.77 Impact Factor -
Article: Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli.
[show abstract] [hide abstract]
ABSTRACT: We have isolated insertion and deletion mutants in glnB, the structural gene of PII, a member of the adenylylation system for glutamine synthetase of Escherichia coli, to study the role of PII in the regulation of the synthesis of glutamine synthetase and of histidase in response to nitrogen deprivation or excess. We have studied the effects of this mutation alone and combined with null mutations resulting from the insertion of transposons or from a deletion in the other genes affecting this regulation, glnD, glnF (ntrA), glnG (ntrC), and glnL (ntrB). Our results confirm that only the products of glnF and glnG are essential for this regulation. In cells of the wild type, the response is mediated by the products of glnD and glnB via the product of glnL. In the condition of nitrogen excess, PII, the product of glnB, appears to convert the product of glnL to a form that prevents the activation of transcription of the structural genes for glutamine synthetase and for histidase by the products of glnF and glnG. During nitrogen deprivation, uridylyltransferase, the product of glnD, is activated by the intracellular excess of 2-ketoglutarate over glutamine and converts PII to PII-UMP and changes the form of the glnL product to one that stimulates the activation of transcription of glutamine synthetase and histidase by the products of glnF and glnG.Journal of Bacteriology 12/1985; 164(2):816-22. · 3.83 Impact Factor
