Browsing by Author "Zeng, Yimeng"

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    Expanding the Mammalian Synthetic Biology Toolbox: Orthogonal Regulators and Gene Circuits for Monitoring Protein Folding and Degradation
    (2019-04-11) Zeng, Yimeng; Segatori, Laura
    Engineering mammalian cells holds great promise for a variety of biomedical applications, ranging from the design of model systems to study complex biological processes underlying human diseases, to the development of cell-based therapies and the production of therapeutic biomolecules. Cell engineering requires sophisticated molecular tools that can interface with cellular systems, but also provide orthogonal functionalities, to achieve precise control over complex gene networks. In an attempt to expand the mammalian synthetic biology toolbox for applications in the study of protein misfolding diseases, my research seeks to design and construct orthogonal gene regulators and genetic circuits to monitor protein folding and degradation. To develop a cell-based sensor for monitoring protein aggregation, I engineered a split transcriptional repressor that links protein aggregation to expression of an easily detectable reporter. I designed a set of two-fragment tetracycline repressor (TetR) variants that can function as transcriptional AND gates in both bacteria and mammalian cells. I built a protein solubility sensor by co-expressing the large “detector” fragment of the split TetR and a small “sensor” fragment fused to the target protein, and demonstrated that protein aggregation can be detected by monitoring complementation between the “detector” and “sensor” fragments. This split TetR represents a novel genetic component that can be used in bacterial as well as mammalian synthetic biology and a much-needed cell-based sensor for monitoring protein conformation in complex cellular environments. To develop a tunable, hysteretic sensor for detection of proteasomal degradation, I built a genetic circuit (Hys-Deg) based on a self-activation loop consisting of a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). Guided by predictive modeling, I demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA that generates the self-activation loop. I also showed that the system can be finely tuned through dosage of tetracycline to calibrate the circuit for detection of desired levels of UPS activation. This study establishes the design rules for building a hysteretic circuit with an autoregulatory feedback loop and provides a synthetic memory module that can be integrated into regulatory gene networks to study and engineer complex cellular behaviors.
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    Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated α-Synuclein
    (Public Library of Science, 2015) Kilpatrick, Kiri; Zeng, Yimeng; Hancock, Tommy; Segatori, Laura; Bioengineering; Biosciences; Chemical and Biomolecular Engineering
    Aggregation of α-synuclein (α-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of α-syn aggregates results from aberrant accumulation of misfolded α-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of α-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated α-syn by inducing autophagic clearance of α-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-β-cyclodextrin promotes autophagic clearance of aggregated α-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of α-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of α-syn aggregates.