brian covello: nsf reu uchiago research presentation
DESCRIPTION
Brian Covello's research presentation on DNA Damage Induced Nuclear Foci was conducted at the University of Chicago under a grant from the National Science Foundation. Information below taken from nsf.gov The Research Experiences for Undergraduates (REU) program supports active research participation by undergraduate students in any of the areas of research funded by the National Science Foundation. REU projects involve students in meaningful ways in ongoing research programs or in research projects specifically designed for the REU program. This solicitation features two mechanisms for support of student research: (1) REU Sites are based on independent proposals to initiate and conduct projects that engage a number of students in research. REU Sites may be based in a single discipline or academic department or may offer interdisciplinary or multi-department research opportunities with a coherent intellectual theme. Proposals with an international dimension are welcome. (2) REU Supplements may be included as a component of proposals for new or renewal NSF grants or cooperative agreements or may be requested for ongoing NSF-funded research projects. Undergraduate student participants in either REU Sites or REU Supplements must be U.S. citizens, U.S. nationals, or permanent residents of the United States. Students do not apply to NSF to participate in REU activities. Students apply directly to REU Sites or to NSF-funded investigators who receive REU Supplements. To identify appropriate REU Sites, students should consult the directory of active REU Sites on the Web at http://www.nsf.gov/crssprgm/reu/reu_search.cfm. EDUCATIONAL OPPORTUNITY This program provides educational opportunities for Undergraduate Students . This program provides indirect funding for undergraduate students to participate in research. To inquire about possible funding opportunities, please contact the organizations that have received awards. (Do not contact NSF.) A directory of active REU Sites, along with contact information, can be found on the Web at http://www.nsf.gov/crssprgm/reu/reu_search.cfm. NSF funds a large number of research opportunities for undergraduate students through its REU Sites program. An REU Site consists of a group of ten or so undergraduates who work in the research programs of the host institution. Each student is associated with a specific research project, where he/she works closely with the faculty and other researchers. Students are granted stipends and, in many cases, assistance with housing and travel. Undergraduate students supported with NSF funds must be citizens or permanent residents of the United States or its possessions. An REU Site may be at either a US or foreign location. By using the web page, Search for an REU Site, you may examine opportunities in the subject areas supported by various NSF units.TRANSCRIPT
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