This study demonstrated the utility of direct tissue MALDI MS in identifying early markers of antibiotic induced nephrotoxicity. Using a histology directed approach, twelve proteins were identified as having statistically significant changes as a result of gentamicin and kanamycin treatment. These proteins can be linked back to proposed mechanisms of toxicity thereby not only supplying additional diagnostic markers but providing mechanistic insight as well. One of the markers identified was a C-terminally truncated ubiquitin termed ubiquitin-t. Though this protein was originally described as an experimental artifact, this work demonstrates for the first time that the C-terminal cleavage of ubiquitin is created by cathepsin B in vivo. The activity of this cleavage was recapitulated and visualized in situ with exogenous ubiquitin and IMS directly from tissue. The addition of a CATB inhibitor on the tissue inhibited the reaction as evidenced by lack of a signal corresponding to the truncated ubiquitin in our MALDI images. Studies done to examine the effect of our sample preparation on the presence of this feature resulted in the determination that this cleavage was occurring in vivo. MALDI images comparing a control animal and one dosed with a CATB inhibitor supported our hypothesis that CATB was cleaving ubiquitin in vivo. Cathepsin B activity assays revealed that the decrease in ubiquitin-t seen with drug treatment was a result of decreased CATB activity, further providing a link between lysosomal enzyme activity and drug toxicity.

The DNA Damage Response (DDR) is an inducible barrier to tumorigenesis at its earliest stages of development. Evidence from cell culture, animal models, and clinical samples reveals DDR activation in response to activation of oncogenes and inactivation of tumor suppressors. These genetic alterations promote replication stress and DNA damage that activates DDR signaling pathways. The DDR restrains the growth of cells with mutated or unstable genomes by preventing cell cycle progression, promoting DNA repair, and inducing senescence or apoptosis. Several genetic alterations that activate DDR pathways have been identified, and recent sequencing efforts indicate hundreds of genes are mutated in breast or colorectal tumors. The heterogenous basis of cancer suggests there are many genes that can contribute to the process of tumorigenesis, and thus an important goal of cancer research is to understand the mechanisms through which genome instability arises and contributes to disease. I identified seventy-four genes with genome maintenance activity using a functional and biologically relevant RNAi screen that monitors the ability of gene-silencing siRNAs to induce DDR activation. Additional analyses identified thirty-five genes that may possess replication-dependent genome maintenance functions, which I confirmed for two genes of interest after further characterization. I demonstrated that DDB1 maintains genome integrity as part of an ubiquitin-ligase complex that mediates the S phase-dependent degradation of the replication-licensing factor CDT1. Proper regulation of CDT1 is critical to prevent re-replication and subsequent double strand break formation that can threaten genome integrity. In collaboration with Xin Xu, I also demonstrated that the genome maintenance function of CINP involves regulation of ATR-dependent DDR signaling. CINP promotes ATR-mediated phosphorylation of CHK1 and maintenance of the G2 checkpoint, in addition to facilitating CDK2-dependent phosphorylation of ATRIP. The seventy-four genome maintenance genes I identified are excellent candidates for the gene function defects that may promote genome instability and DDR activation in pre-cancerous lesions. Further characterization of these genes may provide a greater understanding of the gene functions and cellular pathways that are critical for tumorigenesis.

Genetic instability is a hallmark of cancer. The ATR-ATRIP complex functions at the apex of a cell cycle checkpoint signaling pathway that is critical during every cell division cycle to maintain genome integrity. ATR activity is critical for regulating the firing of replication origins, stabilizing and repairing damaged replication forks, and preventing the premature onset of mitosis. I have developed a model system for identifying checkpoint protein localization to stalled replication forks using CHIP. By stalling replication fork at a defined site on an episome, I have created a system with greater resolution than that conventionally used for protein localization studies. In collaboration with additional lab members, I have also identified a novel ATR signaling regulator, CINP. CINP interacts with ATR-ATRIP and is required for ATR-dependent Chk1 phosphorylation and maintenance of the G2 checkpoint. Finally, I have also examined the recruitment of checkpoint proteins to sites of DNA damage. This recruitment of ATR-ATRIP is mediated by an interaction between the checkpoint recruitment domain (CRD) of ATRIP and the ssDNA binding protein RPA. I found that two other checkpoint proteins, Rad9 and Mre11, contain a similar acid sequence, and that the CRD domains of ATRIP, Rad9, and Mre11, all contact the basic cleft of RPA70N in a similar manner. The ability of Rad9 to relocalize to DNA damage sites is compromised when the RAD9-RPA70 interaction is disrupted. Furthermore, mutations in the Rad9 CRD domain cause hypersensitivity to DNA damage and compromise ATR-dependent CHK1 phosphorylation. Mutations within the RPA70N OB fold impair checkpoint activation, but do not interfere with the DNA replication. My research has resulted in the development of a system for identifying checkpoint proteins recruited to stalled replication forks, identified a novel regulator of ATR signaling, and identified a protein-protein interaction surface within RPA70 that makes contacts with multiple checkpoint proteins to promote ATR signaling.

Phospholipase C-γ1 (PLC-γ1) mediates cell adhesion and migration through an undefined mechanism. Here, we examine the role of PLC-γ1 in cell-matrix adhesion in a hanging drop assay of cell aggregation. Plcg1 Null (-/-) mouse embryonic fibroblasts formed aggregates that were larger and significantly more resistant to dissociation than cells in which PLC-γ1 is re-expressed (Null + cells). Aggregate formation could be disrupted by inhibition of fibronectin interaction with integrins, indicating that fibronectin assembly may mediate aggregate formation. Fibronectin assembly was mediated by integrin α5β1 in both cell lines, while assays measuring fibronectin assembly revealed increased assembly in the Null cells. Null and Null + cells exhibited equivalent fibronectin mRNA levels and equivalent levels of fibronectin protein in pulse-labeling experiments. However, levels of secreted fibronectin in the conditioned medium were increased in Null cells. The data implicates a negative regulatory role for PLC-γ1 in cell aggregation by controlling the secretion of fibronectin into the media and its assembly into fibrils.

p73, a transcription factor that plays essential roles during development and tumor suppression, shares significant sequence identity with p53. However, p53 and p73 are regulated by different pathways, and p73 is not mutated in human tumors. How tumors tolerate over-expression of p73, a protein with tumor suppressive properties, is unclear. We hypothesized that signaling pathways upstream of p73 inhibit its activity in tumors. We devised an approach, based on recognition of patterns within a p73 gene signature, that identified mTOR as an upstream inhibitor of p73. In addition, p73 is a substrate for mTOR and associates with mTOR in cells. mTOR inhibition leads to an elevation in p73 levels and an increase in its activity at target genes involved in apoptosis, metabolism, and autophagy. There is a critical need to understand not only genes and non-coding RNAs regulated by p73, but also how this regulation changes during treatment regimens. We used ChIP-on-Chip to obtain a comprehensive profile of p73 binding sites across the genome: the p73 cistrome. We measured this profile again in the presence of the mTOR inhibitor rapamycin, which did not alter the overall distribution of p73-bound loci but instead selectively increased p73 occupancy at ~9% of all binding sites. Further, we propose an anti-cancer strategy that targets the mTOR-p73 signaling axis as demonstrated in two mesenchymal tumor types: basal-like breast cancers and rhabdomyosarcomas. Using a gene signature of the mTOR-p73 signaling axis, we demonstrated that p73 is functional in a subset of rhabdomyosarcomas and is a determinant of clinical outcome. These data are consistent with the observation that genes and miRNAs directly regulated by p73 exhibit patterns of expression conserved during both mesenchymal differentiation and tumorigenesis, functions that can be explored in mice and in human tumors using new tools that we have developed. Signatures similar to those presented herein might inform the use of cancer therapies such as mTOR inhibitors that engage p73 and are affected by differential p73 activities in tumor subtypes.

In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis, I define a domain within the JM region of the epidermal growth factor receptor (EGFR) that plays an activating role. This region is termed the juxtamembrane activation domain (JMAD). The JMAD encompasses residues 664-682 and is encoded by a portion of exon 18, which also encodes part of the tyrosine kinase domain N-lobe. I describe how an uncharacterized lung cancer mutation within the JMAD (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay. The biochemical, biological, and structural data presented within this dissertation illustrates the importance of the JMAD in EGFR tyrosine kinase activation and inhibition. In a related, but distinct project, I examine the role of the p97 AAA-ATPase in mediating nuclear localization of the EGFR. I demonstrate maximal ligand-dependent association of p97 and EGFR at a time point consistent with a previously described trafficking phenomenon which involves retrotranslocation of mature EGFR to the endoplasmic reticulum and then to the nucleus. Additionally, disruption of ligand-dependent nuclear trafficking of EGFR upon knockdown of p97 is shown. Data presented in this dissertation suggests that p97 is required for ligand-dependent nuclear localization of the EGFR.

The integrity and propagation of the genome depends upon the fidelity of DNA processing events such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation of single-stranded DNA (ssDNA). As the primary eukaryotic ssDNA-binding protein, Replication Protein A (RPA) protects ssDNA templates from stray nuclease cleavage and untimely reannealment. More importantly, RPA serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes. <p> As a universal participant in DNA processing, RPA must interact with a wide array of structurally unique multi-protein complexes and ssDNA substrates. The flexible, modular organization of the protein is thought to be critical for enabling such structural adaptability. Despite the availability of high-resolution x-ray and NMR structures of individual RPA domains, the dynamic interdomain organization of full-length RPA and the accompanying structural alterations imposed by DNA processing have not been extensively characterized. <p> The scope of this dissertation research has focused upon probing the solution arrangement of modular domains within full-length RPA and the rearrangement of this inter-domain architecture as RPA engages ssDNA substrates. NMR studies on the full-length protein resolve conflicting views of RPA architecture within the literature, indicating an absence of inter-domain contacts and favoring a model for flexible independence of RPA domains. NMR 15N relaxation studies on select tandem domain fragments from RPA provide a detailed biophysical description of the inter-domain dynamics indicated by NMR studies on the full-length protein, revealing that the rotational motion of modular domains is largely dependent upon the length of the interconnecting linker, as well as the presence of ssDNA substrate. Results from small-angle x-ray scattering (SAXS) studies on RPA's DNA-binding core provide insight into the inter-domain rearrangements that accompany RPA as it proceeds through its three modes of DNA-binding, suggesting that the DNA-binding core progressively compacts as it proceeds through initial and intermediate binding modes, but loses this compaction upon transitioning to the final binding mode. <p> This work has provided the first view of the global disposition of RPA's inter-domain organization and how RPA's dynamic quaternary structure is refashioned upon binding ssDNA. These findings serve as an essential prerequisite to understanding how RPA coordinates access to ssDNA templates and regulates progression of DNA processing events.

Genome sequencing projects have revealed that glutathione (GSH) transferases are widely distributed in bacteria but most remain only as annotations in sequenced genomes. The goal of this project was to assign function to members of the GSH transferase superfamily from Escherichia coli K-12. Several strategies are required for the accurate assignment of function to proteins of unknown function. These approaches include; 1) analysis using informatics and sequence similarity, 2) co-localization of genes providing operon/metabolic context, 3) transcriptional analysis, 4) phenotypic response to gene knockouts, 5) structural biology, 6) and functional assays of the protein. The work described in this report focuses on functional and structural studies of three GSH transferase homologs from E. coli, YfcG, YghU and YqjG. The YghU and YfcG proteins represent a previously unrecognized class of GSH transferases that have unique structural and catalytic properties. These two proteins have a distinct active site and the ability to bind glutathione disulfide (GSSG) with high affinity (YfcG) or two molecules of GSH simultaneously (YghU). The proteins share robust disulfide-bond oxidoreductase and GSH-dependent peroxidase activity. Despite the similarities, YghU and YfcG have significant chemical and structural differences, including their preferred oxidation states and the N-terminal and C-terminal extensions of YghU. YqjG belongs to a unique cluster of GSH transferases that has not been previously characterized. The protein exhibits modest disulfide bond reductase activity and may have glutathionyl-hydroquinone reductase activity with other substrates. The protein adopts a classical GSH transferase fold but a unique splayed dimer. Proteins in this class are primarily from bacteria, although some are from eukaryotes including fungi, and may constitute a novel class of GSH transferases.

Molecular events involved in successful embryo implantation take place in defined time and space but are not generally well understood. Here for the first time, we use MALDI Imaging Mass Spectrometry (IMS) technologies to elucidate the spatial patterning of lipids and proteins during embryo implantation and demonstrate its value in helping to understand fertility and the reproductive process. For example, our analysis clearly shows previously unknown molecular distributions of lipids over time in this system. Beyond this example, we think that the approach and the concepts illustrated in this work will be of value in various areas of biology and will be critical for understanding many biological processes.

Diepoxybutane is a mutagenic and carcinogenic oxidation product of the important industrial chemical and environmental contaminant butadiene. The mutagenic potential of diepoxybutane is thought to be due in part to its bifunctional electrophilic character. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-protein or peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) or glutathione transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein crosslink formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate due to the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH crosslink formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. The candidate proteins histones H2b and H3 were identified in screens using human liver nuclei and the bis-electrophile 1,2-dibromoethane. Incubation of these proteins with diepoxybutane resulted in DNA-protein cross-links and produced protein adducts, and DNA-histone H2b cross-links were identified (immunochemically) in E. coli cells expressing histone H2b. However, heterologous expression of histone H2b in E. coli failed to enhance bis-electrophile-induced mutagenesis, although histone H2b bound DNA with even higher affinity than AGT. The extent of DNA cross-linking of isolated histone H2b was similar to that of AGT, suggesting that differences in post-cross-linking events explain the difference in mutagenesis. In a related experiment, reactive diepoxybutane-glutathione conjugates believed to contribute to enhanced mutagenesis observed in bacterial cells overexpressing glutathione transferases were investigated. Mass spectral analysis of incubations containing purified glutathione transferase, glutathione, and diepoxybutane yielded a glutathione conjugate that retained the epoxide. Diepoxybutane also produced glutathione-DNA cross-links upon incubation.