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Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies

Nature Methods volume 14, pages 279282 (2017) | Download Citation

Abstract

The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells.

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Acknowledgements

The work was supported by Scuola Normale Superiore institutional funds and by a grant from the European Union Seventh Framework Program (grant no. 604102; Human Brain Project). We are grateful to M.-H. Kuo and D. Guo (Michigan State University, USA) for the H3 tethered catalysis bait, to A. Cereseto (University of Trento, Italy) for the HIV-1 integrase tethered catalysis bait. We acknowledge A. Allouch for helpful discussions about integrase protocols and V. Liverani for help in bait cloning and technical laboratory support. We thank T.H. Rabbitts, J. Zeng, and G. Meli for discussions on the yeast selection procedures. We also thank M. Maffei and R. Ciampi for collaborative logistic help. Many thanks to F. Cremisi and to A. Cellerino for helpful discussions.

Author information

Affiliations

  1. Bio@SNS Laboratory, Scuola Normale Superiore, Pisa, Italy.

    • Michele Chirichella
    • , Simonetta Lisi
    • , Marco Fantini
    • , Martina Goracci
    • , Mariantonietta Calvello
    • , Cristina Di Primio
    •  & Antonino Cattaneo
  2. Genomic Facility, European Brain Research Institute (EBRI), Roma, Italy.

    • Rossella Brandi
    • , Ivan Arisi
    •  & Mara D'Onofrio

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Contributions

A.C. conceived the project; A.C. and M. Chirichella designed the experiments; M. Chirichella performed all the experiments except for the integrase infectivity assay, which was designed, executed, and analyzed by C.D.P.; M. Chirichella and S.L. performed bait validation, Y2H initial setup, and cDNA library construction; M. Chirichella, S.L., and M.F. executed integrase yeast screening; M. Calvello purified and tested intrabody proteins; M. Chirichella and M.G. performed H3 yeast screening; S.L., M. Chirichella, M.D., and R.B. performed microarray experiment; M.F., I.A., M. Chirichella and M.G. analysed microarray data; M.G. performed RT-qPCR experiments; A.C. supervised work; A.C. and M.C. analyzed and discussed the data. M. Chirichella and A.C. wrote the paper with input from all coauthors.

Competing interests

The authors A.C. and M.C. have filed a PCT patent application which includes data and materials described in this manuscript.

Corresponding author

Correspondence to Antonino Cattaneo.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Tables 1–4.

  2. 2.

    Supplementary Protocol

    PISA Supplementary Protocol.

Image files

  1. 1.

    Supplementary Data

    Heat map of the significant differentially expressed genes between scFv112A (anti-acIN) and scFv58F (anti H3AcK9) samples.

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DOI

https://doi.org/10.1038/nmeth.4144

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