• Recombinant NSMCE2 Has Auto-SUMOylation and SUMO-Chain Assembly Activities

      Ellis, Nathan; Longmire, Pierce; Paek, Andrew; Weinert, Ted (The University of Arizona., 2019)
      Homologous recombination (HR) is a high-fidelity repair mechanism that primarily functions during replication-associated DNA damage and plays a major role in preventing genomic instability. As an effective repair mechanism, HR is also postulated to drive tumor cell resistance to platinum-based chemotherapeutic agents that induce DNA damage. RAD51 is an essential protein in the HR pathway, and its overexpression has been associated with chemotherapy-resistant tumors. Thus, RAD51 and the SUMOylation processes involved in its regulation are of interest when looking for ways to combat this resistance. Depletion of the E3 SUMO ligase NSMCE2 inhibits RAD51 function in HR. Further, preliminary studies have identified a candidate compound, C3, that potentially inhibits NSMCE2 activity and increases cancer cell sensitivity to chemotherapy. Through recombinant protein expression in E. coli and affinity chromatography, I obtained partially purified polyhistidine-tagged NSMCE2 protein. This protein exhibits both auto-SUMOylation and SUMO-chain assembly activities in vitro. However, C3 inhibits neither of these activities. Altogether, this study provides insights into the function of NSMCE2 and sets up future studies to investigate the inhibition of NSMCE2 activity.
    • Sensitizing Human Cancer Cells Through the Inhibition of NSMCE2

      Ellis, Nathan; Alassady, Hanen Assad; Weinert, Ted; Paek, Andrew (The University of Arizona., 2018)
      Homologous recombination (HR) is a high-fidelity DNA repair pathway that is activated mainly by replication-associated DNA damage. DNA damaging agents that cause double strand breaks (DSBs) at the replication fork can be repaired by HR. NSMCE2, an E3 sumo ligase, regulates HR at damaged replication forks. NSMCE2, complexed with the structural maintenance of chromosomes (SMC) 5/6 complex, stimulates the SUMOylation of the SMC6 coiled-coil region, thus mediating HR. Cancers that have defects in their HR machinery are susceptible to chemotherapies that elicit DNA damage, and in particular, damage that affects DNA replication. Targeting regulators of HR could therefore sensitize HR-proficient cancers to standard therapies. We used in-silico protein modeling to generate a 3D model of the human NSMCE2 protein. We identified a deep pocket in NSMCE2 that is adjacent to an active-site zinc-binding region and twelve compounds that potentially could occupy that pocket. We sought to determine whether any of these compounds could inhibit NSMCE2 activity or function. We screened the compounds in the human osteosarcoma cell line (U2OS) for synergistic inhibition of cellular proliferation by the topoisomerase 1 inhibitor, camptothecin, and identified four that caused no cellular toxicity on their own whereas they were synergistic with camptothecin in limiting cell proliferation. One of the compounds, compound 3, showed a twelve-fold increase in inhibition of cellular proliferation in combination with camptothecin. We also were able to determine compound specificity to NSMCE2 using a mutation in HEK293T cells. We concluded using small molecule inhibitors of NSMCE2 could be useful in enhancing chemotherapy-mediated killing of HR-proficient cancer cells. Development of NSMCE2 inhibitors could also lead to adjuvant therapies that would allow for patients to receive lower doses of toxic topoisomerase poisons.
    • Upstream Regulators of TORC1 Signaling Pathway in Saccharomyces cerevisiae

      Capaldi, Andrew; Vaishampayan, Prajakta; Capaldi, Andrew; Gutenkunst, Ryan; Paek, Andrew (The University of Arizona., 2019)
      Many nutrients including glucose, phosphate and amino acids regulate TORC1 activity and when the cells are in stress or starvation conditions, TORC1 activity is inhibited. It remains unclear how this happens. So, we are interested in mapping the signaling system that talks to TORC1 to ultimately understand how the complex integrates signals to control growth in stress conditions. In this project, the response of TORC1 under nitrogen, glucose and phosphate starvation conditions was checked in strains lacking one or more stress signaling pathway proteins to identify the mechanism which leads to inhibition of TORC1 in stress conditions.
    • Worm Paparazzi – A High Throughput Lifespan and Healthspan Analysis Platform for Individual Caenorhabditis elegans

      Sutphin, George; Freitas, Samuel; Trouard, Ted; Bilgin, Ali; Paek, Andrew; Sutphin, George (The University of Arizona., 2021)
      Aging is a near ubiquitous phenomenon prevalent across evolutionary lineages from single-celled organisms to humans. Natural aging is characterized by a progressive deterioration in cellular and tissue function. Age is the primary risk factor for the many of the most prevalent diseases and pathological conditions today. Lifespan and healthspan measurements are the gold standard phenotypes for aging research, integrating the influence of many complex molecular processes into a set of easily measurable physiological metrics. Due to the number of environmental, genetic, and phenotypic inputs, a high degree of variation is present between individuals within a given population. The roundworm Caenorhabditis elegans is among the most common model systems used to study aging, because of its short lifespan (~3 weeks), simplicity to culture in the lab, and availability of powerful genetic tools. Lifespan is traditionally measured through population-level survival assays counted by hand on petri plates containing solid nematode growth media (NGM) and seeded with a bacterial food sourec, with each plate containing a sample of a few dozen animals to represent a population. These studies can give an accurate measure of aging within restricted populations under experimental conditions but require many hours of human attention. The amount of resources required to create statistically well-powered data is inefficient in terms of cost, time, and labor. In order to alleviate this research bottleneck, different mechanical systems have been proposed and implemented, each with their own advantages and disadvantages. Robotic systems have been created that can recapitulate manual lifespan results. Most of these systems either rely on population-level assessment of lifespan, require manual input during data processing, or necessitate maintaining worms in a liquid culture environment. Liquid culture causes C. elegans to swim continuously, placing the animals into a stress state that can change the molecular processes that influence aging. Thus, by changing the environmental context, new discoveries in liquid culture cannot be directly compared to the majority of previous work on C. elegans aging conducted on solid culture. Here I describe Worm Paparazzi, a novel analysis system designed to automate lifespan and healthspan measurement for individual roundworms cultured on solid media. This system uses the WorMotel™ (Churgin et al. 2017) a 240-well plate constructed from polydimethylsiloxane (PDMS) that isolates individual C. elegans on solid media under conditions similar to traditional manual lifespan assays. By leveraging the characteristics of the WorMotel™, creating supporting robotics, and building a custom analysis architecture, the Worm Paparazzi analysis system is able to combine the accuracy of human lifespan screeners—while removing one source of human bias—with the high-throughput capabilities of a machine-based system for an individualized insight into C. elegans lifespan and healthspan assays, thereby providing researchers with a new, powerful tool to study the macroscopic result of lifespan and healthspan assays.