BACKGROUND & AIMS:
:Osteopontin (OPN) expression is reduced in surviving dopaminergic neurones in the substantia nigra (SN) in Parkinson's disease (PD), and protects against MPP(+) -induced cell death in primary mesencephalic cultures and 6-OHDA-induced cell loss in the rat, while inactivation of OPN aggravates cell death. OPN is thought to act through interactions with integrin receptors or CD44. However, the specific protein interactions involved in OPN-mediated neuroprotection are unknown and are the focus of this study. The yeast two-hybrid (YTH) technique was utilised to investigate OPN-protein interactions, using full-length human OPN to screen a human foetal brain cDNA library. Proteins involved in apoptosis, protein degradation and microtubule stability were identified as OPN binding partners. These included: MAP1A and MAP1B, which regulate microtubule stability; RNF138, an E3 ubiquitin-ligase; proteasome β1 subunit, a subunit of the 20S proteasome involved in the ubiquitin-dependent cleavage of peptides; BAG6, SGTΑ and EF1A, proteins implicated in control of apoptosis; DnaJB1, a co-chaperone of Hsp70s; and pleiotrophin, a growth factor. The use of site-directed mutagenesis to modify known OPN protein binding sites outside the RGD integrin binding domain, specifically Y165A and D139E, inhibited some of these interactions. Further investigation using affinity pull-down assays, co-immunoprecipitation and immunohistochemistry confirmed that OPN associates with MAP1A and MAP1B in rat SN and striatum. These findings indicate a role for OPN in the regulation of microtubule dynamics, apoptosis and proteolysis in the brain, suggesting that OPN may act as an endogenous multifunctional protective protein in PD.
背景与目标:
:在帕金森病(PD)的黑质(SN)中幸存的多巴胺能神经元中,骨桥蛋白(OPN)的表达减少,并防止MPP()诱导的原发性中脑培养物中的细胞死亡和6-OHDA诱导的中性粒细胞丢失大鼠,而OPN失活会加剧细胞死亡。 OPN被认为是通过与整联蛋白受体或CD44相互作用来发挥作用的。但是,参与OPN介导的神经保护的特定蛋白质相互作用尚不清楚,并且是本研究的重点。酵母双杂交(YTH)技术用于研究OPN-蛋白相互作用,使用全长人OPN筛选人胎脑cDNA文库。参与凋亡,蛋白质降解和微管稳定性的蛋白质被鉴定为OPN结合伴侣。其中包括:MAP1A和MAP1B,它们调节微管的稳定性; RNF138,E3泛素连接酶;蛋白酶体β1亚基,是20S蛋白酶体的一个亚基,参与泛素依赖性肽的裂解。 BAG6,SGTAA和EF1A蛋白与细胞凋亡的控制有关; DnaJB1,Hsp70s的伴侣分子。以及促生长素-促生长素。使用定点诱变来修饰RGD整联蛋白结合域之外的已知OPN蛋白结合位点,特别是Y165A和D139E,抑制了其中某些相互作用。使用亲和力下拉测定,免疫共沉淀和免疫组织化学的进一步研究证实,OPN与大鼠SN和纹状体中的MAP1A和MAP1B缔合。这些发现表明OPN在调节脑中微管动力学,凋亡和蛋白水解中的作用,表明OPN可能是PD中的内源性多功能保护蛋白。