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Collagen Prolyl 4-Hydroxylase as a Therapeutic Target.

ABSTRACT: Collagen is the dominant protein of the extracellular matrix. Its distinguishing feature is a three-stranded helix of great tensile strength. (2 S,4 R)-4-Hydroxyproline residues are essential for the stability of this triple helix. These residues arise from the post-translational modification of (2 S)-proline residues by collagen prolyl 4-hydroxylases (CP4Hs), which are members of the Fe(II)- and ?-ketoglutarate (AKG)-dependent dioxygenase family. Here, we provide a framework for the inhibition of CP4Hs as the basis for treating fibrotic diseases and cancer metastasis. We begin with a summary of the structure and enzymatic reaction mechanism of CP4Hs. Then, we review the metal ions, metal chelators, mimetics of AKG and collagen strands, and natural products that are known to inhibit CP4Hs. Our focus is on inhibitors with potential utility in the clinic. We conclude with a prospectus for more effective inhibitors.

SUBMITTER: Vasta JD

PROVIDER: S-EPMC6344319 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Collagen Prolyl 4-Hydroxylase as a Therapeutic Target.

Vasta James D JD Raines Ronald T RT

Journal of medicinal chemistry 20180723 23

Collagen is the dominant protein of the extracellular matrix. Its distinguishing feature is a three-stranded helix of great tensile strength. (2 S,4 R)-4-Hydroxyproline residues are essential for the stability of this triple helix. These residues arise from the post-translational modification of (2 S)-proline residues by collagen prolyl 4-hydroxylases (CP4Hs), which are members of the Fe(II)- and α-ketoglutarate (AKG)-dependent dioxygenase family. Here, we provide a framework for the inhibition ...[more]

PMID: 29986141

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Project description:BackgroundWe describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H), the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H alpha2beta2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-alpha subunit antibody magnetic beads and an anti-beta subunit antibody binds to the PDI/beta subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation.ResultsWe applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase) verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield.ConclusionsHere we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due to the specificity of the antibodies used in the assay against the different C-P4H subunits, the method detects the entire holoenzyme, and the signal is not disturbed by background expression of the separate subunits.

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Collagen Prolyl 4-Hydroxylase as a Therapeutic Target.

Publications

Collagen Prolyl 4-Hydroxylase as a Therapeutic Target.

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