Discover Of An Oncoprotein And Importance Of Databases

MDMD2 And GenBank

MDM2 is an interesting protein. As You know p53 is a protein for suppressing tumor-like events and an essential protein during the G0 stage in the cell division cycle. It’s about 53 kilodaltons (kDa). The discovery of MDM2 in GenBank is as important as It’s also a p53 inhibitor protein.

Approximately a 90 kDa protein was discovered in 1990 by Hinds et al. To identify the 90 kDa protein, there is something called an affinity column was created (Momand et al., 1992). Affinity columns are used for separating a biomolecule from a mixture in chromatography. The column is made with an antibody that connects p53 protein. The experiment was done by growing the cells in a dish, breaking the cell membrane, and pouring everything into the affinity column. The reason was to have p53 and 90 kDa connected to p53 in the same column. So the complex structure will be captured in that column. The p53 protein and its connected protein, 90 kDa were released from the column by adding a peptide that dissociated the p53 from the column. In the gel, the complex of p53 and 90 kDa was separated from the other molecules. Trypsin is used to digest that 90 kDa protein to turn it into peptides. The primary structures (sequences) of these three peptides determined by Edman degradation sequencing, were: PLLLK, AKLESSDQAEEGLDVPDGK, and VAQMLLSQESDDYSQPSTS.

The researchers used a tool called “tBLASTn” (search translated nucleotide databases using a protein query - BLAST stands for Basic Local Alignment Searching Tools) to identify possible proteins in a database called “GenBank” for a match to the two long peptide sequences above. The tool takes the genetic code to translate all the DNA sequences in GenBank to amino acid sequences and compares our sequences to them. Interestingly when the researchers attempted the first time, there were no matches found. However, They tried again in GenBank yet one month later, surprisingly GenBank contained a match! The result was a gene that had just been deposited to GenBank by the scientist “Donna George” (University of Pennsylvania).

The GenBank annotations of Donna George’s gene told us which it coded for a protein promoted tumor growth. The identical match between the 90 kDa protein and the gene showed that the gene protein product, MDM2, and the 90 kDa protein were the same. Furthermore, It suggested that MDM2 causes cancer by binding to and inhibiting p53 tumor suppressor activity. Later, MDM2 is found to modify p53 with ubiquitin, which ultimately targets p53 for degradation. This snippet of research history shows the importance of GenBank and scientific discovery.

References

1. Fakharzadeh, S. S., Trusko, S. P., & George, D. L. (1991). Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J, 10(6), 1565–1569.
2. Hinds, P. W., Finlay, C. A., Quartin, R. S., Baker, S. J., Fearon, E. R., Vogelstein, B., & Levine, A. J. (1990). Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the “hot spot” mutant phenotypes. Cell Growth Differ, 1(12), 571–580.
3. Momand, J., Zambetti, G. P., Olson, D. C., George, D., & Levine, A. J. (1992). The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69(7), 1237–1245.