Background | Projects

Projects

• Systems Toxicology of Chemotherapeutic Alkylating Agents
• Input and Output Modulators of DNA Repair
• Damage Induced Signaling Pathways

Systems Toxicology of Alkylating Agents

The cyotoxicity of alkylating agents influences efficacy of some chemotherapeutic regiments, yet most of the genetic modulators of alkylating agent induced cell death are still unknown. A major focus of my laboratory is to use high throughout approaches to analyze and characterize the toxicity of alkylating agents. We have previously used global data sets, in conjunction with computational techniques, to identify important biological responses that modulate cytotoxicity. Specifically, we have performed high throughput phenotyping assays and developed a number of computational techniques to identify activated protein networks and protein complexes that modulate the toxicity of alkylating agents. Falling under the auspice of Systems Toxicology, these global techniques utilize in silico methods to build pathways and model cellular responses to pharmacologically important compounds. The development of computational techniques and data analysis approaches is applicable to other biological perturbations, and provides fundamental principles for pathway analysis in the fledgling fields of Systems Biology and Systems Toxicology.

Figure 1. Computational approaches that combine high throughput data sets were used to identify activated networks, pathways and complexes that prevent damaged induced cell death. Shown here (left) is high throughput phenotypic data mapped to protein-protein and protein-DNA interactions. Mathematical analysis of mapped data (right) was then used to identify highly connected structures that are heavily enriched for protein complexes and signaling pathways. These systems biology based approaches represent an in silico method for piecing together cellular pathways.

Input and Output Modulators of DNA Repair

Ascertaining susceptibility factors that predispose cells towards DNA damage induced death and carcinogenesis is a goal of our research. In pursuit of this, a major focus is to characterize enzyme systems and protein-protein interactions that coordinate the repair of DNA alkylation damage. Different DNA repair pathways all possess the potential to repair DNA alkylation damage. These pathways differ with respect to fidelity, thus, making the cellular decision-making process and subsequent direction of repair key elements to outcome. We believe that cellular decision making and ensuing direction of repair requires a high degree of coordination at the protein level, via protein-protein interactions that direct repair into the proper pathway and protein-protein interactions that preserve the fidelity of each pathway once started. We are therefore testing the hypothesis that human proteins that interact with key DNA repair pathways are critical for preventing alkylation-induced cytotoxicity.

Figure 2. Molecular interactions represent a basic cellular approach for guiding pathways and stimulating biochemical reactions. Using steady state kinetic analysis (top) we have shown that DNA repair proteins working alone are not as efficient as two working together, and that a DNA repair activity can be stimulated by the presence of other connected proteins. A DNA repair network (bottom panel) based on a single protein (in the center) and other proteins that interact with it by protein-protein interactions (blue line) can be constructed and the influence of accessory proteins on thermodynamic and kinetic parameters of the reaction can be tested, to help understand the molecular basis of the network.

Damage Induced Signaling Pathways

Cell stress initiated by alkylating agents can lead to the activation and deactivation of many cellular processes via small molecule signaling to nucleic acids; which include DNA, mRNA, rRNA, tRNA and nucleotide pools. We are currently investigating a novel damage induced signaling mechanism through tRNA, that appears to translationally regulate damage response proteins. Damage signaling via tRNA modifications has the potential to translationally regulate cohesive groups of proteins in response to damage and we are performing targeted biochemical and genetic experiments to analyze this hypothesis.

Figure 3. 1-methyladene and 3-methylcytosine represent the methylated forms of adenine and cytosine and are some of the methylated bases that occur in DNA and RNA. It should be noted that methylation can occur by random chemical events and also by enzymatic transfer reactions.

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