Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma

J Biol Chem. 2021 Dec;297(6):101353. doi: 10.1016/j.jbc.2021.101353. Epub 2021 Oct 27.

Abstract

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.

Keywords: Nore1A; RASSF5; Ras effector protein; Ras inhibitors; Ras oncogene; protein engineering; protein-protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • A549 Cells
  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics*
  • Adaptor Proteins, Signal Transducing / metabolism
  • Adenocarcinoma of Lung / genetics*
  • Adenocarcinoma of Lung / metabolism
  • Adenocarcinoma of Lung / pathology
  • Apoptosis Regulatory Proteins / chemistry
  • Apoptosis Regulatory Proteins / genetics*
  • Apoptosis Regulatory Proteins / metabolism
  • Genes, Tumor Suppressor
  • Humans
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism
  • Lung Neoplasms / pathology
  • Models, Molecular
  • Mutation
  • Protein Binding
  • Protein Domains
  • ras Proteins / genetics
  • ras Proteins / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • RASSF5 protein, human
  • ras Proteins