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Determining the Structure of Oncogenic NRas Mutants
Author(s) -
Ojeda Monica,
Reid Derion,
Mattos Carla
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.619.18
Subject(s) - gtpase , mutant , neuroblastoma ras viral oncogene homolog , gtp' , guanine nucleotide exchange factor , hras , guanosine , signal transduction , biology , microbiology and biotechnology , guanosine triphosphate , gene isoform , biochemistry , mutation , chemistry , gene , enzyme , kras
Ras proteins are small GTPases that are involved in signal transduction pathways that regulates cell proliferation, differentiation, and survival. These proteins act as molecular switches where they are activated by exchanging guanosine diphosphate (GDP) to guanosine triphosphate (GTP), and become inactivated when GTP is hydrolyzed to GDP. Ras GTPases are mutated in 20% of human cancers. Within the Ras family, there are three isoforms: H‐Ras, K‐Ras, and N‐Ras. Due to the high structural similarities of the three isoforms, much of the structural efforts have focused on H‐ and K‐Ras while neglecting N‐Ras. However, it is known that N‐Ras oncogenes are predominantly mutated in melanoma. Two common sites of oncogenic mutations that affect N‐Ras regulation of cancerous pathways are Gly12 and Gln61. Mutants at codons 12 and 61 result in constitutive activation. Therefore, we are interested in using x‐ray crystallography to solve the structure of N‐Ras oncogenic mutants: G12D and G12S. Both mutants are highly expressed in E. coli and behave well through our established purification protocol involving ion exchange and gel filtration chromatography. Both were purified in sufficiently high yield for structural biology studies and are currently undergoing crystallization trials. We expect that mutating glycine 12 will interfere with nucleotide binding and inhibit hydrolysis of N‐Ras leading to constitutive activation. By determining the structure of these oncogenic N‐Ras mutants, better therapeutic targets can potentially be developed. Support or Funding Information National Science Foundation, Northeastern University