Buschdorf Jan Paul, Chew Li Li, Soh Unice Jim Kim, Liou Yih-Cherng, Low Boon Chuan
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Republic of Singapore.
PLoS One. 2008 Jul 16;3(7):e2686. doi: 10.1371/journal.pone.0002686.
Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation.
编码脑特异性蛋白BNIP-H（或Caytaxin）的ATCAY基因突变会导致开曼小脑共济失调。BNIP-H与谷氨酰胺酶结合，谷氨酰胺酶是一种产生神经递质的酶，并影响其活性和细胞内定位。在此，我们描述了BNIP-H与肽基脯氨酰顺/反异构酶Pin1之间结合的鉴定和特征。在神经生长因子刺激后，BNIP-H与Pin1相互作用，它们在分化的嗜铬细胞瘤PC12细胞和胚胎癌P19细胞的神经突和细胞质中共定位。缺失诱变揭示了BNIP-H C末端内的两个隐蔽结合位点，影响Pin1 WW结构域的单点突变完全消除了它们的结合。尽管这两个位点不包含任何典型的Pin1结合基序，但它们对Pin1 WW结构域突变体S16E、S16A和W34A以及其异构酶结构域的催化惰性C113A显示出不同的结合谱。此外，它们的直接相互作用仅在通过两个相似区域破坏BNIP-H形成分子内相互作用的能力时才会发生。此外，在神经生长因子刺激下，Pin1的表达破坏了PC12细胞中BNIP-H/谷氨酰胺酶复合物的形成。这些结果表明，神经生长因子可能通过释放其分子内抑制来刺激BNIP-H与Pin1的相互作用。这种机制可以在神经元分化过程中对BNIP-H的细胞活性提供翻译后调控。
