Discovery Molecular Pharmacology Department, Discovery Screening Center, Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho,Aoba-ku, Yokohama 227-0033, Japan.
J Biol Chem. 2012 Feb 24;287(9):6810-8. doi: 10.1074/jbc.M111.280248. Epub 2012 Jan 10.
Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.
热休克蛋白 47（Hsp47）是一种胶原特异性分子伴侣，对于各种类型前胶原的成熟至关重要。先前的研究表明，Hsp47 可能优先识别内质网中前胶原的三螺旋形式，而不是展开的前胶原链。然而，由于目前用于检测 Hsp47 与胶原体外和体内相互作用的方法存在局限性，其潜在机制仍不清楚。在这项研究中，我们通过采用体外时间分辨荧光共振能量转移（FRET）技术和体内双分子荧光互补技术，为此建立了新的方法。使用这些方法，我们提供了直接的证据，证明 Hsp47 在体外与胶原三螺旋结合，而不是与单体形式结合。我们还证明了 Hsp47 在体内与三螺旋构象的胶原模型肽结合。Hsp47 不与体内修饰以阻止其形成三螺旋的胶原肽结合。这些结果明确表明 Hsp47 在体外和体内识别前胶原的三螺旋形式。
