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A BRET-based assay reveals collagen-Hsp47 interaction dynamics in the endoplasmic reticulum and small-molecule inhibition of this interaction.

ABSTRACT: Molecular chaperones perform pivotal roles in proteostasis by engaging in protein-protein interactions (PPIs). The collagen-specific molecular chaperone Hsp47 (heat shock protein 47) interacts with procollagen in the endoplasmic reticulum (ER) and plays crucial roles in collagen synthesis. PPIs between Hsp47 and collagen could offer a therapeutic target for fibrosis, which is characterized by abnormal collagen accumulation in the extracellular matrix of fibrotic organs. Herein, we established a bioluminescence resonance energy transfer (BRET) system for assessing Hsp47-collagen interaction dynamics within the ER. After optimization and validation of the method, we could demonstrate inhibition of the interaction between Hsp47 and collagen by a small molecule (Col003) in the ER. Using the BRET system, we also found that Hsp47 interacts not only with the Gly-Pro-Arg motif but also weakly with Gly-Pro-Hyp motifs of triple-helical collagen in cells. Moreover, we found that the serpin loop of Hsp47 (SerpinH1) contributes to its binding to collagen. We propose that the method developed here can provide valuable information on PPIs between Hsp47 and collagen and on the effects of PPI inhibitors important for the management of fibrotic disorders.

SUBMITTER: Ito S

PROVIDER: S-EPMC6827286 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A BRET-based assay reveals collagen-Hsp47 interaction dynamics in the endoplasmic reticulum and small-molecule inhibition of this interaction.

Ito Shinya S Saito Masazumi M Yoshida Masahito M Takeuchi Koh K Doi Takayuki T Nagata Kazuhiro K

The Journal of biological chemistry 20190906 44

Molecular chaperones perform pivotal roles in proteostasis by engaging in protein-protein interactions (PPIs). The collagen-specific molecular chaperone Hsp47 (heat shock protein 47) interacts with procollagen in the endoplasmic reticulum (ER) and plays crucial roles in collagen synthesis. PPIs between Hsp47 and collagen could offer a therapeutic target for fibrosis, which is characterized by abnormal collagen accumulation in the extracellular matrix of fibrotic organs. Herein, we established a ...[more]

PMID: 31492754

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Project description:Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time-course over which these studies were conducted, we cannot assign causal primacy to either the effects on hepatic lipid homeostasis or ER stress responses.

Dataset Information

A BRET-based assay reveals collagen-Hsp47 interaction dynamics in the endoplasmic reticulum and small-molecule inhibition of this interaction.

Publications

A BRET-based assay reveals collagen-Hsp47 interaction dynamics in the endoplasmic reticulum and small-molecule inhibition of this interaction.

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