Investigating Minor States of the Oncoprotein N-MYC, with Focus on Proline Cis/Trans Isomerisation using NMR Spectroscopy

Sammanfattning: MYC is a family of three regulator genes that codes for transcription factors. Expression of Myc proteins from MYC genes is found to be deregulated in 70 % of all cancer forms. The three human homologs C-Myc, N-Myc and L-Myc are mainly associated with cancer in the lymphatic system, nerve tissues and lung cancer, respectively. Even though N-Myc is associated with Neuroblastoma, the cancer variant that is most common among children, the field is focused towards C-Myc. The activation of C-Myc begins with phosphorylation of Serine 62, followed by trans-to-cis isomerisation of Proline 63. Then Threonine 58 becomes phosphorylated leading to that Serine 62 is dephosphorylated and subsequent cis-to-trans isomerisation of Proline 63, and C-Myc is marked for degradation. Cis-trans isomerisation is necessary for regulation of gene expression, and is therefore important to understand. Since N-Myc and C-Myc have identical sequences between residues 47 to residue 69, the hypothesis is that N-Myc is activated in the same manner, but this has not been confirmed. In this project the first 69 amino acids of N-Myc were analysed with NMR spectroscopy. This resulted in a near complete assignment of the major conformation, and of the alternative minor conformations as well. The traditional assignment experiments HNCACB, HN(CO)CACB, HNCO, HN(CA)CO in combination with CCH-TOCSY and HN(CCO)C revealed that the majority of the minor configurations can be explained by cis/trans isomerisation of prolines. In addition, the protein was analysed with direct carbon detected NMR spectroscopy to be able to detect the prolines.