Rice University

Events at Rice

Thesis Defense

Graduate and Postdoctoral Studies
BioSciences-Biochemistry and Cell Biology

Speaker: Yiwen Liu
Masters Candidate

Optimization of the Proteasomal Degradation Reporter (eDeg-On) System for CRISPR-mediated Whole-genome Knockout Screens

Wednesday, April 12, 2017
10:00 AM  to 12:00 PM

801  BioScience Research Collaborative

Protein folding and clearance of misfolded proteins are crucial to maintain cellular homeostasis (Jariel-Encontre et al., 2008). Misfolded proteins may associate with other cellular components and possibly impair their functions. They may also self-associate to form insoluble aggregates, which are the hallmarks of a number of neurodegenerative diseases, such as Parkinson’s (Olanow and McNaught, 2006) and Alzheimer’s (Oddo, 2008). The ubiquitin proteasome system (UPS) is the main pathway that catalyzes the degradation of soluble misfolded proteins in mammalian cells. Therefore, enhancing the UPS activity through activation of the proteasome is considered as a promising strategy to ameliorate phenotypes associated with the accumulation of misfolded proteins. However, our current understanding of the molecular mechanism underlying proteasomal degradation is still limited, and this lack of understanding limits the rational design of pharmacologic strategies to increase UPS activity. As a result, proteasome activators are rare and remain poorly characterized (Huang and Chen, 2009). Some evidence suggests that modulation of specific components of the UPS results in increased degradation of target proteins (Rechsteiner and Hill, 2005 and Vilchez et al., 2012), but the exact mechanism of action remains to be elucidated. To detect proteasome activation without understanding the underlying mechanism, an orthogonal genetic circuit (the eDeg-On system) was devised in my laboratory to monitor changes in UPS activity. This circuit links an increase in UPS activity to an increase in fluorescent output. To introduce genetic manipulation at the whole-genome scale, the CRISPR-cas technology has emerged as powerful tool to achieve easy and precise genetic modifications both at the single-gene level and at the whole-genome scale, and can be adapted to introduce mutations to components of the UPS. I optimized the eDeg-On system and evaluated it for pooled screening of whole-genome CRISPR-mediated knockout library. I replaced the antibiotic resistance gene in the eDeg-On system (now named as eDeg-On_2 system), and assessed the response of HEK293 cells stably expressing the eDeg-On_2 system to modulation of proteasomal degradation. To evaluate the use of the HEK293/eDeg-On_2 stable cell line as a reporter assay in the context of a pooled CRISRP-mediated screen, I conducted mock screens using different ratios of positive and negative controls. I concluded that the eDeg-On_2 system is robust and can be used as a reporter assay for CRISPR-mediated whole-genome knockout screens. The use of the eDeg-On_2 system in combination with the CRISPR-cas technology to identify proteasome regulators will contribute to the development of therapeutic strategies for protein misfolding diseases. Further applications include targeting the UPS function for therapeutic applications as well as for enhancing the production of recombinant proteins in industrial settings.

<<   June 2017   >>
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30

Search for Events