Modification Of H-Ras Research Articles

Epitranscriptic regulation of HRAS by N6-methyladenosine drives tumor progression

Overexpression of Ras, in addition to the oncogenic mutations, occurs in various human cancers. However, the mechanisms for epitranscriptic regulation of RAS in tumorigenesis remain unclear. Here, we report that the widespread N6-methyladenosine (m6A) modification of HRAS, but not KRAS and NRAS, is higher in cancer tissues compared with the adjacent tissues, which results in the increased expression of H-Ras protein, thus promoting cancer cell proliferation and metastasis. Mechanistically, three m6A modification sites of HRAS 3' UTR, which is regulated by FTO and bound by YTHDF1, but not YTHDF2 nor YTHDF3, promote its protein expression by the enhanced translational elongation. In addition, targeting HRAS m6A modification decreases cancer proliferation and metastasis. Clinically, up-regulated H-Ras expression correlates with down-regulated FTO and up-regulated YTHDF1 expression in various cancers. Collectively, our study reveals a linking between specific m6A modification sites of HRAS and tumor progression, which provides a new strategy to target oncogenic Ras signaling.

Photocontrol of Small G-Protein H-RAS Multimer Formation using Caged Compounds

The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switching mechanisms utilizing conformational changes in the nucleotide-binding motifs have been well studied at the molecular level. H-Ras has the hypervariable region (HVR) at the C-terminal, in which three cysteine residues are located. It is known that HVR is the site of lipid modification; farnesylation and palmitoylation. Lipid modification enable H-Ras bind to membrane to act physiological role. Recently we have observed that the chemical modification of the cysteine residues in the HVR with hydrophobic thiol reactive reagents induces multimer formation of H-Ras. In this study, utilizing the phenomenon, we attempted to control the transition from multimer to monomer of H-Ras by light irradiation. We employed thiol reactive caged-compound, 2-Nitrobenzyl bromide (NBB). NBB was incorporated into the cysteine residues in HVR stoichiometrically. The modification of H-Ras with NBB induced formation of multimer. We monitored the transition between the monomer and multimer by Size-exclusion chromatography coupled with high-performance liquid chromatography (SEC-HPLC). Light irradiation at 400 nm induced elimination of 2-Nitrobenzyl group, resulting in conversion of multimer to monomer. The elution pattern of the multimer on SEC-HPLC indicated the multimer is composed of five Ras molecules. Small angle X-ray scattering (SAXS) analysis of the multimer also suggested that the NBB modified Ras formed the pentamer. The global shape of the multimer was deduced from the data of SAXS. We prepared H-Ras mutants which intrinsic cysteine residues in HVR were substituted with serine in order to identify which cysteine residue is essential for the multimerization. Incorporations of NBB into more than two residues among the three cysteines were shown to be necessary for multimerization.