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

Anamika Singh,Ariel Erijman,Ashish Noronha,Hemant Kumar,Yoav Peleg,Yosef Yarden,Julia M Shifman

doi:10.1016/j.jbc.2021.101353

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.

Highlights

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record
Proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish pro-cancer pathways initiated by Ras oncogene
Using a combination of computational design and in vitro evolution, we engineered highaffinity Ras-binding proteins starting from a natural Ras effector, Ras Association Domain Family 5 (RASSF5) (NORE1A), which is encoded by a tumor suppressor gene

Summary

Results

Design of a focused RASSF5 library RASSF5 utilizes 15 residues to interact with Ras. In principle, we could randomize all of these residues to select the best binders to Ras through directed evolution. We decided to design a focused library of RASSF5 mutants to limit randomization to only most promising positions and amino acids For this purpose, we first performed computational saturation mutagenesis of the RASSF5/Ras-GTP complex [45], where all the binding interface residues on RASSF5 were mutated to 17 amino acids. We designed a single RASSF5 library containing variants with improved binding to both Ras-GTP and Ras-GDP, while limiting its size to 1.7 x 106 variants to allow for full assessment of all variants with the Journal Pre-proof YSD technology (Figure 1C). Tyrosine at position 234 is the most selected amino acid, reaching 100% for GTP-Ras-selected clones and about 50% in the GDP-selected clones, with tryptophan being another option Both mutations are predicted to improve packing between the two proteins. At position 305, WT methionine is substituted by an arginine that could create salt bridges with D30 and D31 on Ras (Figure 4D)

Binding affinity measurements

Discussion

Engineering by Combined Computational and In Vitro Evolution