Zanata Silvio M, Lopes Marilene H, Mercadante Adriana F, Hajj Glaucia N M, Chiarini Luciana B, Nomizo Regina, Freitas Adriana R O, Cabral Ana L B, Lee Kil S, Juliano Maria A, de Oliveira Elizabeth, Jachieri Saul G, Burlingame Alma, Huang Lan, Linden Rafael, Brentani Ricardo R, Martins Vilma R
Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109 4A, São Paulo 01509010, Brasil.
EMBO J. 2002 Jul 1;21(13):3307-16. doi: 10.1093/emboj/cdf325.
Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.
朊病毒由一种名为PrP(c)的正常唾液酸糖蛋白的异构体组成，其生理作用一直在研究中，重点是筛选配体。我们的团队借助针对由互补亲水性推导的肽段的抗体，描述了一种66 kDa的膜结合PrP(c)蛋白。通过在二维蛋白质凝胶的蛋白质印迹中使用这些抗体，随后对特定斑点进行测序，我们现在已将该分子鉴定为应激诱导蛋白1（STI1）。我们发现该蛋白除了在细胞质中存在外，在细胞膜上也有发现。这两种蛋白以特定且高亲和力的方式相互作用，解离常数K(d)为10(-7) M。相互作用位点被定位到PrP(c)的113 - 128位氨基酸和STI1的230 - 245位氨基酸。细胞表面结合和下拉实验表明重组PrP(c)与细胞内的STI1结合，共免疫沉淀实验强烈表明这两种蛋白在体内相互关联。此外，PrP(c)与STI1或与我们发现的代表STI1结合域的肽段相互作用会诱导神经保护信号，从而使细胞免于凋亡。
