Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments
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Kristina Mayer
Abstract
We hypothesized that tissue-specific expression of cathepsin B-enhanced green fluorescent protein (CB-EGFP) can be driven by the A33-antigen promoter that contains positive cis-regulatory elements, including caudal-related homeobox (CDX) binding sites. The intestine-specific transcription factor Cdx1 is crucial for A33-antigen promoter activation and could thereby induce expression of CB-EGFP. This concept was tested by construction of the vector pA33-CathB-EGFP encoding CB-EGFP downstream of the A33-antigen promoter. Its Cdx1 dependence, as an indication of its intestine-specific expression, was tested in Cdx1-negative CHO-K1 cells. Cdx1 expression was achieved upon transfection with pCdx1-DsRed-Express and was indicated by red fluorescence of the simultaneously translated reporter protein. Immunolabeling with Cdx1-specific antibodies showed correct targeting of the transcription factor to its point of action in nuclei of transfected cells. Co-transfection experiments with plasmids pA33-CathB-EGFP and pCdx1-DsRed-Express confirmed the hypothesis that Cdx1 indeed activates CB-EGFP expression in a manner dependent on the A33-antigen promoter. Co-localization with compartment-specific markers and subcellular fractionation confirmed CB-EGFP trafficking along the expected route to endolysosomal compartments. Hence, the A33-antigen promoter represents a potent tool for induction of Cdx1-dependent CB-EGFP expression in vitro. Our proof-of-principle studies confirm the suitability of this approach in visualizing protease transport in Cdx1-positive tissues of the gastrointestinal tract.
©2008 by Walter de Gruyter Berlin New York
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- Editorial
- Farewell to Hans Fritz, Executive Editor
- Guest Editorial
- Highlight on Advances in Proteolysis Research
- Highlight: 5th General Meeting of the International Proteolysis Society 2007
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- Alternative pathways for production of β-amyloid peptides of Alzheimer's disease
- Bauhinia Kunitz-type proteinase inhibitors: structural characteristics and biological properties
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- Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments
- Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display
- In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells
- Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer
- Regulation of cathepsin K activity by hydrogen peroxide
- Protein Structure and Function
- Contribution of the C30/C75 disulfide bond to the biological properties of onconase
- Conformational changes in bovine lactoferrin induced by slow or fast temperature increases
Articles in the same Issue
- Editors' Note
- Editors' Note
- Editorial
- Farewell to Hans Fritz, Executive Editor
- Guest Editorial
- Highlight on Advances in Proteolysis Research
- Highlight: 5th General Meeting of the International Proteolysis Society 2007
- Proteinases as hormones: targets and mechanisms for proteolytic signaling
- Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution
- Alternative pathways for production of β-amyloid peptides of Alzheimer's disease
- Bauhinia Kunitz-type proteinase inhibitors: structural characteristics and biological properties
- Angiotensin-converting enzyme limits inflammation elicited by Trypanosoma cruzi cysteine proteases: a peripheral mechanism regulating adaptive immunity via the innate kinin pathway
- How Na+ activates thrombin – a review of the functional and structural data
- Cancer cells, adipocytes and matrix metalloproteinase 11: a vicious tumor progression cycle
- Isoaspartate residues dramatically influence substrate recognition and turnover by proteases
- Isoaspartate-containing amyloid precursor protein-derived peptides alter efficacy and specificity of potential β-secretases
- Trial of the cysteine cathepsin inhibitor JPM-OEt on early and advanced mammary cancer stages in the MMTV-PyMT-transgenic mouse model
- Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17
- Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments
- Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display
- In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells
- Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer
- Regulation of cathepsin K activity by hydrogen peroxide
- Protein Structure and Function
- Contribution of the C30/C75 disulfide bond to the biological properties of onconase
- Conformational changes in bovine lactoferrin induced by slow or fast temperature increases
