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Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB

Nature Structural & Molecular Biology volume 20, pages 495–501 (2013)Cite this article

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Abstract

The neuronal isoform of cytoplasmic polyadenylation element–binding protein (CPEB) is a regulator of local protein synthesis at synapses and is critical in maintaining learning-related synaptic plasticity in Aplysia. Previous studies indicate that the function of Aplysia CPEB can be modulated by conversion to a stable prion-like state, thus contributing to the stabilization of long-term memory on a molecular level. Here, we used biophysical methods to demonstrate that Aplysia CPEB, like other prions, undergoes a conformational switch from soluble α-helix–rich oligomer to β-sheet–rich fiber in vitro. Solid-state NMR analyses of the fibers indicated a relatively rigid N-terminal prion domain. The fiber form of Aplysia CPEB showed enhanced binding to target mRNAs as compared to the soluble form. Consequently, we propose a model for the Aplysia CPEB fibers that may have relevance for functional prions in general.

Although significant knowledge of cellular and molecular mechanisms underlying the acquisition and early storage of implicit and explicit long-term memory has been gained, the mechanisms by which memories are maintained for long periods of time are still not fully understood1,2. Because proteins normally have relatively short half-lives, of hours or days, the question remains: How can the change in molecular composition of a synapse be maintained for long periods of time, as is required for long-term memory? We previously found one answer to this conundrum in a work describing a prion-like regulator of local protein synthesis at the synapse in the marine snail Aplysia californica: the cytoplasmic polyadenylation element–binding protein Aplysia CPEB3,4. This provided physiological evidence that the prion-like properties of Aplysia CPEB might explain the self-sustained, continuous molecular turnover at the synapse5.

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Figure 1: Schematic representation of the neuronal isoform of Aplysia CPEB.
Figure 2: The soluble form of Aplysia CPEB exists as oligomers in Aplysia neurons and in E. coli expressing Aplysia CPEB.
Figure 3: RNA-binding assay.
Figure 4: Secondary-structure analysis of two conformational forms of full-length Aplysia CPEB.
Figure 5: X-ray powder diffraction analyses of Aplysia CPEB fibers.
Figure 6: 1D solid-state NMR spectra of the fiber form of full-length Aplysia CPEB and the isolated prion domain, indicating dynamic RNA-binding domains and static prion domains.
Figure 7: 2D solid-state NMR spectra on the N-terminal prion-like domain of Aplysia CPEB.

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Acknowledgements

We are extremely grateful to K. Si (Stowers Institute for Medical Research, Kansas City, Missouri, USA) for providing the plasmids used in this study and to S. Jokusch (Columbia University) for helping us with FTIR spectra and real-time fluorescence measurement. We would like to acknowledge and thank J. Lidestri, (Columbia University) and H. Park (Scripps Research Institute) for assisting with X-ray diffraction analyses of the fibers. We are grateful to K. Nakanishi and N. Berova (Columbia University) for their guidance in CD analysis and to M.A. Gawinowicz (Columbia University) for MS analysis of the protein fragments. This work was supported by the Howard Hughes Medical Institute, US National Institutes of Health (grant MH075026; E.R.K.) and US National Science Foundation (grant MCB0316248; A.E.M.).

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Author notes

  1. Bindu L Raveendra, Ansgar B Siemer & Sathyanarayanan V Puthanveettil

    Present address: Present addresses: Department of Chemistry, Scripps Research Institute, Jupiter, Florida, USA (B.L.R.), Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, California, USA (A.B.S.) and Department of Neuroscience, Scripps Research Institute, Jupiter, Florida, USA (S.V.P.).,

Authors and Affiliations

  1. Department of Neuroscience, Columbia University, New York, New York, USA

    Bindu L Raveendra, Sathyanarayanan V Puthanveettil & Eric R Kandel

  2. Department of Chemistry, Columbia University, New York, New York, USA

    Ansgar B Siemer & Ann E McDermott

  3. Howard Hughes Medical Institute, Columbia University, New York, New York, USA

    Sathyanarayanan V Puthanveettil, Wayne A Hendrickson & Eric R Kandel

  4. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Wayne A Hendrickson & Eric R Kandel

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Contributions

B.L.R., S.V.P. and A.B.S. designed the project after discussing with E.R.K., A.E.M. and W.A.H. All the experiments were done by B.L.R. except NMR and RNA-binding experiments. NMR experiments were done by A.B.S. and RNA-binding experiments by S.V.P. B.L.R., A.B.S., E.R.K. and A.E.M. wrote the paper.

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Correspondence to Eric R Kandel or Ann E McDermott.

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Raveendra, B., Siemer, A., Puthanveettil, S. et al. Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB. Nat Struct Mol Biol 20, 495–501 (2013). https://doi.org/10.1038/nsmb.2503

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