Biverstål Henrik, Dolfe Lisa, Hermansson Erik, Leppert Axel, Reifenrath Mara, Winblad Bengt, Presto Jenny, Johansson Jan
Karolinska Institutet, Department of NVS, Center for Alzheimer Research, Division of Neurogeriatrics, 141 57 Huddinge, Sweden.
Karolinska Institutet, Department of NVS, Center for Alzheimer Research, Division of Neurogeriatrics, 141 57 Huddinge, Sweden.
Biochim Biophys Acta. 2015 Aug;1854(8):835-43. doi: 10.1016/j.bbapap.2015.04.005. Epub 2015 Apr 17.
The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid β-peptide (Aβ) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer. We show here increased dissociation of the BRICHOS trimer into monomers, by addition of detergents or of bis-ANS, known to bind to the putative substrate-binding site, or by introducing a Ser to Arg mutation expected to interfere with trimer formation. This leads to increased capacity to delay Aβ(42) fibril formation. Cross-linking of the BRICHOS trimer with glutaraldehyde, in contrast, renders it unable to affect Aβ(42) fibril formation. Moreover, proSP-C BRICHOS expressed in HEK293 cells is mainly monomeric, as detected by proximity ligation assay. Finally, proteolytic cleavage of BRICHOS in a loop region that is cleaved during proSP-C biosynthesis results in increased capacity to delay Aβ(42) fibril formation. These results indicate that modulation of the accessibility of the substrate-binding site is a means to regulate BRICHOS activity.
BRICHOS结构域与人类淀粉样疾病相关，并且在体外和体内均能有效阻止淀粉样β肽（Aβ）形成淀粉样纤维。通过X射线晶体学、分析超速离心、非变性聚丙烯酰胺凝胶电泳、分析尺寸排阻色谱和电喷雾质谱观察发现，重组人表面活性蛋白C（proSP-C）的BRICHOS结构域形成同源三聚体。据推测，该三聚体是一种无活性的储存形式，因为在单体中鉴定出的一个假定底物结合位点埋藏在三聚体的亚基界面中。我们在此表明，通过添加已知可结合假定底物结合位点的去污剂或双-ANS，或通过引入预期会干扰三聚体形成的丝氨酸到精氨酸突变，BRICHOS三聚体解离为单体的程度增加。这导致延迟Aβ（42）纤维形成的能力增强。相比之下，用戊二醛对BRICHOS三聚体进行交联使其无法影响Aβ（42）纤维形成。此外，通过邻近连接分析检测发现，在HEK293细胞中表达的proSP-C BRICHOS主要为单体形式。最后，在proSP-C生物合成过程中被切割的环区域对BRICHOS进行蛋白水解切割，导致延迟Aβ（42）纤维形成的能力增强。这些结果表明，调节底物结合位点的可及性是调节BRICHOS活性的一种方式。
