This thesis concentrates on understanding how individual nonspecific DNA–protein contacts are used in the excision mechanism of the human DNA repair enzyme, alkyladenine DNA glycosylase (AAG). Initially, studies focus on understanding the structure and magnitude of these fundamentally different DNA–protein stacking and T-shaped interactions to be applied to the active site of AAG. High-level ab initio techniques revealed fundamental knowledge about the structure and magnitude of these distinctly different – and +– contacts between (one or two) conjugated amino acid(s) and one nucleobase. Additionally, the mechanism used by AAG to excise (neutral and cationic) damaged nucleotides was investigated using a hybrid ONIOM approach. Reaction potential energy surfaces reveal that AAG prefers to excise both neutral and cationic substrates through a concerted mechanism, yet the nonspecific contacts present in the active site are only catalytic for the cleavage of the neutral substrates. / xvi, 195 leaves : ill. (some col.) ; 29 cm + 1 CD-ROM

Indiana University-Purdue University Indianapolis (IUPUI) / Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.