Event Title

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

Disciplines

Biology

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


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Apr 28th, 8:50 AM Apr 28th, 9:15 AM

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