Browsing Senior Honors Theses by Subject "X-Ray Crystallography"
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Functional Analysis of Wild-Type LGN and T450 MutantsThe protein LGN, named for the many repeats of the amino acids leucine (L), glycine (G) and asparagine (N), is crucial for mammalian cellular division. Specifically, LGN plays a significant role in cell polarity and alignment of the mitotic spindles and in its absence, the organism ceases to develop. In breast cancer, LGN is upregulated due to phosphorylation of T450 and the knockdown of LGN activity has been shown to suppress growth of breast cancer cells. Furthermore, a mutation to alanine at the 450th position also suppressed breast cancer cell growth. The goal of this project was to explore the biochemistry of both wild-type LGN and two T450 mutants of LGN in hope of gaining insight as to how LGN phosphorylation proliferates cancer. LGN was transiently expressed in BHK-570 tissue culture cells using a pCMV-LGN plasmid and LipoD 293 reagent. Protein expression of wild-type LGN was confirmed by both Western blot and immunocytochemistry of fixed, permeabilized cells using anti-GPSM2 antibody. Furthermore, the T450A and T450D mutants, which mimic the unphosphorylated and phosphorylated forms of LGN respectively, are being generated by PCR and will be sent for sequencing analysis to confirm the completed mutagenesis. Future experiments will determine the effect of phosphorylation of T450 on LGN function; specifically, additional localization studies of T450A and T450D mutants will be conducted and compared to that of wild-type LGN. Characterization of LGN function relative to the phosphorylation status of T450 could lead to the development of novel treatments for breast cancer.