Brian Covello: NSF REU UChiago Research Presentation
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Transcript of Brian Covello: NSF REU UChiago Research Presentation
Proximity Proteomics of DNA Damage Induced Nuclear Foci
Brian Covello The Kron Lab
Proximity Proteomics • APEX Technology – First uClized as EM tag, monomeric 28kDa
– Ascorbate Peroxidase that oxidizes phenol derivaCves to radicals • Living cellular protein-‐protein interacCons (PPIs)
• Short lived (<1 msec) • Small labeling radius (~20nm) • Radicals covalently react with Trp, Tyr, His, Cys (invesCgate weak/transient interacCons)
– BioCnylaCon is a rare protein modificaCon
APEX Advantages • AcCve in all cellular
compartments (unlike HRP)
• Insoluble proteins/Membrane Proteins
• Natural seXng • RegulaCon of
bioCnylaCon process (H2O2)
• Weak or transient interacCons (<1msec)
Disadvantages • Some proteins, such as
various histones, are endogenously bioCnylated
• False negaCves • Specificity
Goals
• ValidaCon of APEX technology – TargeCng to mitochondria à mito-‐APEX
• CreaCon of APEX template vector within pTRIO • Gene specific APEX fusions (53BP1, RAD51) • Proximity proteomics of various subcellular compartments – Mitochondria, transmembrane, ER
• Proteomic environment in non-‐senescent v. senescent cells, DNA damage response
ValidaCon of APEX
• APEX reacCon • BioCn-‐tyramide (500 μM, 1 mM, 2.5 mM, 30 min) • H2O2, 1 min, 3 micromolar • Quench reacCon (anC-‐oxidants)
• Expression of APEX (anC-‐V5), bioCnylaCon (streptavidin-‐HRP)
Mito-‐APEX Expression
Mito-‐APEX bioCnylaCon
Immunofluorescence Microscopy
APEX Fusions
DNA Damage Response
Conclusions • Mito-‐APEX expressed, targeted to mitochondria, and bioCnylates in MCF7 cells (V5)
• APEX expression unaffected by bioCn-‐tyramide concentraCons
• 60 and 80 kDa bioCnylated bands previously reported are present our control lanes
• BioCnylaCon is increased as concentraCon of bioCn-‐tyramide is increased
• pTRIO-‐V5-‐APEX template successfully engineered • pTRIO-‐V5-‐APEX-‐53BP1 successfully engineered
Future • Mapping of subcellular compartment proteins by mass spectrometry
• ConstrucCon of pTRIO-‐V5-‐APEX-‐RAD51 • Stable expressions à lenCviral • Proximity dependent proteomics of DNA damage response pathway
• Proteomic analysis in pre-‐senescent v. senescent cells
• Enhancing of APEX technology (cell cycle specific?)
Thank You
• Stephen Kron and Oliver Appelbe for mentorship and guidance
• Andy Truman for cloning assistance • NSF REU MGCB at UChicago for financial support