Investigating Nanoluciferase as a Potential Proteome Stress Sensor
Session Number
C07
Advisor(s)
Sue Fox, Northwestern University Richard Morimoto, Northwestern University Anan Yu, Northwestern University
Location
B-125 Tellabs
Start Date
28-4-2016 8:50 AM
End Date
28-4-2016 9:15 AM
Abstract
Firefly luciferase (Fluc) is a well-documented proteome stress sensor. Its misfolding upon cellular stress leads to aggregation and loss of enzymatic activity, which can be assayed by oxidizing luciferin, producing bioluminescence. However, Fluc is large (63 kilodaltons) and dependent on adenosine triphosphate (ATP) for activity. Since stresses to the cell can affect ATP production, Fluc activity is not solely dependent on its proper folding. We examined a luciferase synthesized from deep sea shrimp, nanoluciferase (NanoLuc); smaller (19 kDa), more luminescent than Fluc, and ATP- independent, to determine if it can accurately detect changes in chaperone mediated protein folding upon stress. We used four forms of NanoLuc: wild type, 8M, R116N, and 8M+R116N, with each mutation structurally destabilizing the protein. We expressed the proteins in human neuroblastoma HTB11 cells to examine whether NanoLuc can detect acute stress. We also purified the NanoLuc proteins expressed in Escherichia coli (E. coli) in order to determine if it is in vitro refolding dependent on the 70 kDa heat shock cognate protein (Hsc70). Our preliminary results show that two-hour heat stress led to aggregation of the NanoLuc protein in the cytosol of the cell, suggesting that NanoLuc could behave like a proteome stress sensor
Investigating Nanoluciferase as a Potential Proteome Stress Sensor
B-125 Tellabs
Firefly luciferase (Fluc) is a well-documented proteome stress sensor. Its misfolding upon cellular stress leads to aggregation and loss of enzymatic activity, which can be assayed by oxidizing luciferin, producing bioluminescence. However, Fluc is large (63 kilodaltons) and dependent on adenosine triphosphate (ATP) for activity. Since stresses to the cell can affect ATP production, Fluc activity is not solely dependent on its proper folding. We examined a luciferase synthesized from deep sea shrimp, nanoluciferase (NanoLuc); smaller (19 kDa), more luminescent than Fluc, and ATP- independent, to determine if it can accurately detect changes in chaperone mediated protein folding upon stress. We used four forms of NanoLuc: wild type, 8M, R116N, and 8M+R116N, with each mutation structurally destabilizing the protein. We expressed the proteins in human neuroblastoma HTB11 cells to examine whether NanoLuc can detect acute stress. We also purified the NanoLuc proteins expressed in Escherichia coli (E. coli) in order to determine if it is in vitro refolding dependent on the 70 kDa heat shock cognate protein (Hsc70). Our preliminary results show that two-hour heat stress led to aggregation of the NanoLuc protein in the cytosol of the cell, suggesting that NanoLuc could behave like a proteome stress sensor