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Diverting a protein from its cellular location by intracellular antibodies. The case of p21Ras

M Lener1, I R Horn, A Cardinale

  • 1Department of Neuroscience, University of Rome 'Tor Vergata', Roma, Italy.

European Journal of Biochemistry
|February 15, 2000
PubMed

Insights

New single-chain variable fragments (scFv) targeting Ras proteins were developed using phage libraries. These anti-Ras scFv inhibited cell proliferation when expressed intracellularly, suggesting a broad utility for antibody-based intracellular inhibition strategies.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Immunology

Background:

  • Ras proteins are key regulators of cell signaling pathways.
  • Dysregulation of Ras signaling is implicated in various cancers.
  • Developing tools to inhibit Ras function intracellularly is crucial for cancer research.

Purpose of the Study:

  • To generate novel single-chain variable fragments (scFv) against Ras proteins using phage display technology.
  • To evaluate the intracellular inhibitory potential of these anti-Ras scFv antibodies in mammalian cells.
  • To explore the mechanism of action for intracellular Ras inhibition by these antibody fragments.

Main Methods:

  • Phage display libraries were used to select and derive anti-Ras scFv antibodies.
  • In vitro biochemical assays assessed the interference of scFv with Ras GTPase activity and Ras-Raf binding.
  • Intracellular expression of anti-Ras scFv in mouse 3T3 K-Ras transformed cells.
  • Cell proliferation was measured using bromodeoxyuridine incorporation.
  • Confocal microscopy and double immunofluorescence were employed to determine subcellular localization of scFv and Ras.

Main Results:

  • A panel of anti-Ras scFv antibodies was generated.
  • These scFv did not neutralize Ras function based on in vitro biochemical assays.
  • Intracellular expression of anti-Ras scFv significantly inhibited cell proliferation in Ras-transformed cells.
  • Anti-Ras scFv fragments colocalized with endogenous Ras at aberrant subcellular locations.
  • The intracellular inhibition mechanism appears to involve the diversion of Ras protein traffic.

Conclusions:

  • Phage-derived anti-Ras scFv antibodies can effectively inhibit Ras function and cell proliferation when expressed intracellularly.
  • The in vivo inhibitory capacity of anti-Ras scFv is a frequent property, extending beyond in vitro biochemical neutralization.
  • Intracellular antibody expression offers a versatile strategy for inhibiting protein function, even through non-canonical mechanisms like protein traffic diversion.

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