Ecotin: Lessons on survival in a protease‐filled world

McGrath, Mary E.; Gillmor, S.A.; Fletterick, Robert J.

doi:10.1002/pro.5560040201

Cited by 41 publications

(21 citation statements)

References 38 publications

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“…These binding sites can be fine-tuned by site-directed mutagenesis to potently and specifically inhibit a specific serine protease [6][7][8]. In this work, we have evaluated the ecotin variants as affinity chromatography reagents as they inhibit proteases in the low nanomolar to subnanomolar range, suggesting the formation of stable complexes.…”

Section: Resultsmentioning

confidence: 99%

“…These enzymes may be inhibited by ecotin, a periplasmic Escherichia coli-derived protein and a "fold-specific" inhibitor that has an unusually broad specificity to proteases such as trypsin, chymotrypsin, elastase, factor Xa, kallikrein, and factor XIIa [6,7]. This inhibitor uses two distinct binding sites to recognize its target and has been engineered to potently and specifically inhibit proteases such as urokinase-type plasminogen activator (u-PA) [6][7][8]. Literature described that ecotin is able to purify recombinant trypsinogen expressed in E. coli [9] although it does not recognize more specific proteases such as u-PA.…”

Section: Introductionmentioning

confidence: 99%

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Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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“…It is a periplasmic protein, and is believed to be secreted by invading bacteria during infection in order to inhibit host proteases of the innate immune response such as neutrophil elastase 4 . Ecotin exists in solution as a non-covalent homodimer in which two elongated, 18 kDa monomers associate in a head to tail fashion, mediated by hydrogen bond interactions between residues 125 and 142 in the C-terminal protruding tails of the monomers (Figure 1) 5,6 . This gives the dimer two essentially identical ends, both of which can interact with proteases.…”

Section: Introductionmentioning

confidence: 99%

The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability

McCrudden

Ryan

Turkington

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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The Escherichia coli protease inhibitor ecotin is believed to be implicated in the evasion of host defenses during infection. The protein has also attracted attention as a scaffold for the design of novel, specific protease inhibitors. Ecotin interacts with its targets through two sites. Key hydrophobic residues in both sites (Leu-64, Trp-67, Tyr-69, Met-84, and Met-85) were mutated to alanine and the effects on the inhibition of trypsin, chymotrypsin, and elastase were assessed. Each of these mutant ecotin proteins tested in kinetic assays with these enzymes exerted less inhibitory potency compared to wild-type ecotin. However, these effects were relatively small and not additive. Keywords: Protease inhibitor; trypsin; chymotrypsin; elastase; free energy of interaction; alanine substitution

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“…Another newly identified ModE target, ybhK, encodes a homologue of YvcK, which is required for a normal cell shape and is involved in carbon metabolism in Bacillus subtilis, and restores the growth of the B. subtilis yvcK mutant (38). eco encodes a periplasmic protein of 142 amino acids that is a potent inhibitor of serine protease (39). The ynfEFGHI operon is induced under anaerobic conditions in a fumarate nitrate reduction regulator (FNR)-dependent manner (40).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Set of Genes, Including Nonannotated morA , under the Direct Control of ModE in Escherichia coli

et al. 2013

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dModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5= untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.T he transition metal molybdenum is essential for life. In nature, molybdenum is present in various oxidation states and transported into organisms in the form of the tetraoxyanion molybdate. In the case of Escherichia coli, molybdate is transported through an ABC-type transport system encoded by the modABC operon (1). The expression of the modABC operon is repressed by the molybdate-bound ModE transcription factor (2). The ModE protein functions as a homodimer and consists of two domains, the N-terminal DNA-binding domain containing a winged helixturn-helix motif and the C-terminal molybdate-binding domain (3). Binding of molybdate to the C-terminal domain induces a conformational change of ModE so that it recognizes a palindromic sequence of its target promoters (4). In addition to repression of the molybdate transporter operon, the ModE-molybdate complex is involved in induction of three operons encoding molybdate-containing enzymes, dimethyl sulfoxide (DMSO) reductase (dmsABC), nitrate reductase (napFDAGHBC), and molybdenum cofactor synthase (moaABCDE) (5-7). The three known ModE-inducing targets are all involved in molybdenum metabolism and utilization. Since bacteria contain more than 50 species of the molybdate-containing enzyme, the repertoire of regulation targets of ModE could include more than the already-characterized operons.In this study, attempts were made to identify the set of regulation targets of the E. coli ModE transcription factor. For this purpose, we employed the genetic systematic evolution of ligands by exponential enrichment (SELEX) screening system, which was developed for identification of the set of regulation targets recognized by DNA-binding transcription factors and was successfully used for the search of regulation targets by AscG (8), AllR (9), CitB (10), Cra (11, 12), cyclic AMP receptor protein (CRP) (13), Dan (14), LeuO (15), NemA (16), PdhR (17), RcdA (18), PgrR (19), RstA (20), RutR (21), and TyrR (22). After the genomic SELEX screening, we identified at least 10 binding sites of the ModEmolybdate complex. Transcription in vivo of the predicted promoters located next to ...

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Ecotin: Lessons on survival in a protease‐filled world

Cited by 41 publications

References 38 publications

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

The contribution of key hydrophobic residues in ecotin to enzyme-inhibitor complex stability

Identification of the Set of Genes, Including Nonannotated morA , under the Direct Control of ModE in Escherichia coli

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