In Alzheimer's disease, cytochrome c-dependent apoptosis is a crucial pathway in neuronal cell death. Although beta-amyloid (Aβ) oligomers are known to be the neurotoxins responsible for neuronal cell death, the underlying mechanisms remain largely elusive. Here, we report that the oligomeric form of synthetic Aβ of 42 amino acids elicits death of HT-22 cells. But, when expression of a bcl-2 family protein BAK is suppressed by siRNA, Aβ oligomer-induced cell death was reduced. Furthermore, significant reduction of cytochrome c release was observed with mitochondria isolated from BAK siRNA-treated HT-22 cells. Our in vitro experiments demonstrate that Aβ oligomers bind to BAK on the membrane and induce apoptotic BAK pores and cytochrome c release. Thus, the results suggest that Aβ oligomers function as apoptotic ligands and hijack the intrinsic apoptotic pathway to cause unintended neuronal cell death.

Nucleolar phosphoprotein 140 (Nopp140) is a nucleolar protein, more than 80% of which is disordered. Previous studies have shown that the C-terminal region of Nopp140 (residues 568–596) interacts with protein kinase CK2α, and inhibits the catalytic activity of CK2. Although the region of Nopp140 responsible for the interaction with CK2α was identified, the structural features and the effect of this interaction on the structure of Nopp140 have not been defined due to the difficulty of structural characterization of disordered protein. In this study, the disordered feature of Nopp140 and the effect of CK2α on the structure of Nopp140 were examined using single-molecule fluorescence resonance energy transfer (smFRET) and electron paramagnetic resonance (EPR). The interaction with CK2α was increased conformational rigidity of the CK2α-interacting region of Nopp140 (Nopp140C), suggesting that the disordered and flexible conformation of Nopp140C became more rigid conformation as it binds to CK2α. In addition, site specific spin labeling and EPR analysis confirmed that the residues 574–589 of Nopp140 are critical for binding to CK2α. Similar technical approaches can be applied to analyze the conformational changes in other IDPs during their interactions with binding partners.

Gli3 interact with SPOP. AUC supports a binding model in which the MATH domain can interact with three binding motifs on Gli3-90 with macroscopic weak affinities while FA allows the determination of microscopic affinities. In vitro ubiquitination assays and protein stability studies in mammalian cells with a series of Gli3 SBC motif mutants are used for functional interpretation of our biophysical data. Each SBC motif contributes to SPOP binding in agreement with their similar weak affinities. These results will allow the distinction between a dynamic complex, a static multivalent binding mechanism and large oligomeric assemblies in which many Gli3s co-operate to recruit multiple SPOP dimers. The presence of multiple dispersed SBC motifs suggests their contribution to spatial and temporal control of Gli3 levels in response to Hh signaling. of. Protein kianse CK2 is a ubiquitous kinase that can phosphorylate more than hundreds of cellular proteins, and has important roles in cell growth and development. The interaction of the catalytic subunit of CK2 (CK2alpha) with inositol hexakisphosphate (IP6) and an intrinsically disordered protein, Nopp140, has been analyzed to elucidate the IP6 and Nopp140-dependent regulation mechanism of CK2. X-ray crystallography analysis of the complex of CK2alpha and IP6 showed that lysine rich domain of CK2alpha which locates near the active site was important for the binding to IP6. One of the interaction site of Nopp140 to CK2alpha was identified at the amino acid residues 560~580 by measuring the interactions between the peptides representing different regions of Nopp140. Particularly, the phosphorylation at Ser568 of Nopp140 significantly enhanced its interaction with CK2alpha. These results suggested a regulatory model of Nopp140 and IP6 on CK2alpha in which CK2alpha activity is inhibited by Nopp140 and re-activated by IP6 by competitive binding at the substrate recognition site of CK2alpha. 1205-Pos Board B97Biophysical and Structural Study of Intrinsically Disordered Protein (IDP), Nopp140 Human Nopp140 is a highly phosphorylated nucleolus protein and involved in the biogenesis of the nucleolus. It interacts with a variety of proteins related to the synthesis and assembly of the ribosome including a ubiquitous protein kinase CK2 which mediates cell growth and prevents apoptosis. We showed that hNopp140 is a highly intrinsically disordered protein (IDP) lacking stable secondary structures over its entire sequence of 710 residues. In this work, we employed various biophysical approach, for example, Circular Dichroism (CD), Electron Paramagnetic Resonance (EPR) and Förster resonance energy transfer (FRET), to investigate the intrinsic nature of hNopp140 and its structural changes by various conditions, interaction with CK2alpha. 1206-Pos Board B98Structural Studies of Cytoplasmic P53 Interactions Besides its well-known transcriptional regulatory functions, the tumor suppressor p53 exhibits pro-apoptotic activities in the cytoplasm that are less completely understood. We are undert...