Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer’s Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold–denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloidstructures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores.
Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport, however, the underlying regulatory mechanism is unclear. Here, we identified ICK kinase localization at the tip of cilia as a regulator of ciliary transport. In ICK-deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK-deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT-A, IFT-B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT-B but not IFT-A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.
Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetery (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation enabling the comparison of protein folding, amyloid fibrillation and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.
Shoji Maeda, So Nakagawa, Michihiro Suga, Eiki Yamashita, Atsunori Oshima, Yoshinori Fujiyoshi & Tomitake Tsukihara
Nature 458, 597-602 (2009)
Y. Yonezawa, K. Nakata, K. Sakakura, T. Takada, H. Nakamura
J. Ame. Chem. Soc. (2009) 131 (12), 4535-4540.
A.R. Kinjo, H.Nakamura
Structure (2009) 17 (2), 234-246
Ozawa D, Yagi H, Ban T, Kameda A, Kawakami T, Naiki H, Goto Y
J. Biol. Chem. (2009) 284(2), 1009-1017
Hideaki Tanaka, Koji Kato, Eiki Yamashita Tomoyuki Sumizawa, Yong Zhou, Min Yao, Kenji Iwasaki, Masato Yoshimura, Tomitake Tsukihara
Science 323, 384-388 (2009)
Miyazaki N, Uehara-Ichiki T, Xing L, Bergman L, Higashiura A, Nakagawa A, Omura T, Cheng RH
J. Virol. (2008) 82(22), 11344-11353