In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells
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Sinisa Urban
Abstract
Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.
©2008 by Walter de Gruyter Berlin New York
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- 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
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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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- How Na+ activates thrombin – a review of the functional and structural data
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- 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
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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
