Alterations in the Primary Structures of Ribosomal Proteins in Acquired Drug Resistance
Lohnes, Karen Lynn
Fenselau, Catherine C
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Acquired drug resistance is a multifactorial process that is one of the major causes for cancer treatment failure. The anticancer drug, mitoxantrone, was recently determined to inhibit ribosome biogenesis. Changes in ribosomal protein composition and efficiency with which the ribosomes incorporate 35S-methionine has been noted in a mitoxantrone resistant MCF7 cell line when compared with a drug-susceptible parental cell line. This dissertation evaluated three proteomic workflows in order to successfully characterize the changes in the primary structures of cytoplasmic ribosomal proteins isolated from a mitoxantrone resistant breast cancer cell line that could serve some functional significance to the resistance when compared with a parental drug-susceptible cell line. A combination of the data from the three workflows allowed for the identification of 76 of the 79 human ribosomal proteins with an average sequence coverage of 76%. The N-terminal ends of 52 of the ribosomal proteins were identified using bottom-up and middle-down mass spectrometric approaches. An additional 7 N-terminal fragments were identified by top-down high resolution mass spectrometric analysis. Forty of the 52 N-terminal peptides were observed to have lost their N-terminal methionine and 19 were acetylated. Identification of the N-terminal peptides was most successful using the middle-down approach. Internal acetylations (on lysine) and phosphorylations were only noted with trypsin in-gel digestion and HPLC fraction analysis. Gel arrays of the two ribosomal populations illustrated differences in the protein compositions. Comparative densitometry imaging software indicated the presence of two novel protein spots in the drug resistant cell line as well six additional spots with increased and decreased abundances. High coverage bottom-up mass spectrometric analysis allowed for these protein spots to be assigned as isoform pairs of RPS3, RPS10, RPL11 and RPL23A. Molecular masses and top-down analyses were used to define the alterations in the ribosomal proteins in conjunction with high coverage bottom up and middle-down analyses. The change in the primary structures of these four ribosomal proteins is believed to alter access to the mRNA tunnel in the ribosome. This suggests that these ribosomes may participate in differential selective translation to allow for the cell to produce the necessary proteins during cellular stress.