Complete and accurate DNA replication is essential to maintain the genetic integrity in all organisms. In eukaryotes, the minichromosome maintenance (MCM) complex forms the catalytic core of the CMG helicase that unwinds DNA at the replication fork. We have previously identified a conserved MCM complex binding protein (MCM-BP) through a proteomic screen in human cells. In chapter two of this thesis, I show that MCM-BP makes an important contribution to nuclear morphology in human cells by affecting centrosome duplication. I also show that MCM-BP depletion results in G2 checkpoint signaling and the induction of replication stress. A recent study in Xenopus egg extracts has suggested that MCM-BP functions to unload the MCM complex from chromatin during S-phase. However, the mechanism of this process remains enigmatic. In chapter three of this thesis, I show that MCM-BP directly binds the de-ubiquitylating enzyme, USP7 and that this interaction is mediated by S158 on MCM-BP and the USP7 TRAF domain. Furthermore, I indicate a novel role for USP7 in DNA replication that involves unloading of the MCM complex during S-phase. Finally, my data suggest that MCM-BP tethers an interaction between the USP7 and the MCM complex to facilitate MCM complex unloading at the end of S-phase.

The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt iv activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.

The role of antioxidants in cancer has been controversial for a long time. Although the public’s belief is that antioxidants prevent and/or inhibit cancer, there is increasing evidence to suggest the opposite: that cancer cells require antioxidants to survive. We wanted to interrogate the role of antioxidants in cancer by investigating both upstream regulators and downstream effectors of antioxidant signaling. We have identified protein tyrosine phosphatase non-receptor type 12 (PTPN12) as a novel regulator of antioxidant signaling in cancer. PTPN12 reduces reactive oxygen species (ROS) levels by promoting activity of the forkhead box O (FOXO) family of antioxidant transcription factors. We have also elucidated the impact of glutathione (GSH), the most abundant antioxidant in the cell, on tumorigenesis. We have found that GSH is required for cancer initiation, yet dispensable once transformation has occurred due to compensation provided by the thioredoxin (TXN) antioxidant pathway. Together, these studies expand our knowledge of the role of antioxidants in cancer and provide numerous avenues of research for the future.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.

Understanding the structural basis of specificity and promiscuity of paralogous enzymes is important for deciphering molecular mechanisms and is a necessary step towards designing enzyme-specific modulators. The main objective of this thesis is to provide structural insights that relate protein local sequences to their observed binding and activity profiles through the study of two human protein families – cytosolic sulfotransferases (SULTs) and sirtuins (SIRTs). This was achieved by comparing the family-wide ligand binding fingerprints of these two enzyme families with the structural details of their corresponding enzyme-ligand co-crystal structures. The hSULT enzyme family was profiled against a focused library through binding and activity assays. This suggested a number of novel compounds that bind to the less well-characterized SULT members (SULT1C3 and SULT4A1), and revealed additional broad-spectrum hSULT inhibitors. Based on the profiling data, three enzyme/co-factor/ligand complex structures were solved using X-ray crystallography. The structure of SULT1C2•PAP(3'-phosphoadenosine 5'-phosphate)•pentacholorphenol(PCP) provided a rationale for a novel SULTs inhibition mechanism that depends on substrate acidity. The SULT1B1•PAP•resveratrol structure suggested that the hydrogen-bonding coordination of the 5-OH group on resveratrol is the structural determinant for the observed substrate preference towards resveratrol. SULT2A1•PAP•lithocholic acid(LCA) ternary complex structure confirms that the specificity of SULT2A1 for lithocholic acid derives from its high hydrophobicity in the substrate binding pocket. The same approach was used to interrogate the interaction of the sirtuins with their peptide substrates. The binding and enzymatic assays for human sirtuins have suggested that SIRT1 and SIRT2 are generally less discriminate against substrates while class IV sirtuins - SIRT6 and SIRT7 might be highly specific enzymes. Three different biochemical and kinetic assays showed that SIRT6-dependent histone deacetylation is about 1,000 times slower than for other highly active sirtuins. To understand the molecular basis for the specificity and low activity of SIRT6, I determined the first set of crystal structures for SIRT6 in complex with ADPr (ADP ribose) and the non-hydrolyzable analog of OAADPr (2’-O-acetyl-ADP ribose) – NAADPr (2’-N-acetyl-ADP ribose). The structures revealed human SIRT6 has unique structural features including a splayed zinc-binding domain, lacks a helix bundle and the conserved, highly flexible, NAD(+)-binding loop, which contribute to its observed biochemical behavior.

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Chemotherapeutic agents act by targeting rapidly dividing cancer cells. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. The work presented in this thesis aims to address the basic biology that underlies these issues through the development and application of chemical genomic tools to probe mechanisms of current and novel anticancer compounds. Chemical genomic screens in the yeast Saccharomyces cerevisiae have been used to successfully identify targets and pathways related to a compound’s mode of action. I applied these screens to examine the mode of action of potential anticancer drugs: a class of platinum-acridine compounds and the apoptosis-inducing compound elesclomol. By analogy to the yeast screens, I developed an RNAi-mediated chemical genomic screen in human cells which has the potential to reveal novel targets and drug mechanisms. This screen was applied to further understand doxorubicin’s mode of action. In parallel with the loss-of-function assays, our lab developed a human ORF overexpression screen in human cells. I applied this gain-of-function screen to identify those genes that, when overexpressed, are toxic to cells. Characterization of such genes that cause toxicity can provide insight into human diseases where gene amplification is prevalent.

Drosophila Fat is a large atypical cadherin molecule. Genetic assays show that Fat has multiple function, however, the mechanism of Fat function is poorly understood. Hence, I undertook a biochemical approach to determine the mechanistic function of Fat. Previous data indicated that Fat might be processed; I further confirmed the precursor-product relationships between these proteins. I then looked at sub cellular localization of Fat. My preliminary data suggests that the smaller 110 kDa forms of Ft goes to the nucleus. To characterize the interaction between Fat and Atro, only known binding partner of Fat, I conducted pull-down assays that indicate Fat has multiple binding sites for Atro. However, the interaction is weak, and different experimental conditions will be needed to characterize the interaction. The only known downstream target of both Fat and Atro in PCP is four-jointed. I provided evidence that fjlacZ1.2kb is regulated by the Ecdysone receptor.

Lentivirus-mediated gene therapy has curative potential for a variety of disorders, however, insertional oncogenesis still remains a concern. One approach to increase safety of such treatment modalities is to include a ‘cell fate control safety cassette’ in lentiviral vectors (LVs), enabling pharmacological control over the survival of gene-modified cells (GMCs). Two novel LVs with engineered expression of truncated cell surface molecules (CD19 or LNGFR) fused to a ‘cell fate control’ gene (TmpkF105YR200A) were constructed. Results demonstrated these safety cassettes could be used to control the survival of GMCs in a murine xenogeneic leukemia models. For treatment of Fabry disease, a bicistronic LV containing the fusion safety element and therapeutic α-galactosidase A was constructed. Transduction with this vector restored enzyme activity in Fabry patient’s fibroblasts. These collective results demonstrate that this approach is sufficient to eradicate GMCs, and when combined with a corrective cDNA can provide therapeutic benefit for Fabry disease.