nsf reu uchicago dna damage response poster by brian covello
DESCRIPTION
Brian Covello's science poster details the DNA damage response pathway and proximity proteomics. This project was conducted at the University of Chicago under a grant from the National Science Foundation More information on proteomics below is taken from wikipedia.org Proteomics is the large-scale study of proteins, particularly their structures and functions.[1][2] Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term proteomics was first coined in 1997[3] to make an analogy with genomics, the study of the genome. The word proteome is a blend of protein and genome, and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.[4][5] The proteome is the entire set of proteins,[4] produced or modified by an organism or system. This varies with time and distinct requirements, or stresses, that a cell or organism undergoes. Proteomics is an interdisciplinary domain formed on the basis of the research and development of the Human Genome Project;[citation needed] it is also emerging scientific research and exploration of proteomes from the overall level of intracellular protein composition, structure, and its own unique activity patterns. It is an important component of functional genomics. While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically used for protein purification and mass spectrometry. Complexity of the problem[edit] After genomics and transcriptomics, proteomics is the next step in the study of biological systems. It is more complicated than genomics because an organism's genome is more or less constant, whereas the proteome differs from cell to cell and from time to time. Distinct genes are expressed in different cell types, which means that even the basic set of proteins that are produced in a cell needs to be identified. In the past this phenomenon was done by mRNA analysis, but it was found not to correlate with protein content.[6][7] It is now known that mRNA is not always translated into protein,[8] and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present. Post-translational modifications[edit] Not only does the translation from mRNA cause differences, but many proteins are also subjected to a wide variety of chemical modifications after translation. Many of these post-translational modifications are critical to the protein's function. Phosphorylation[edit] One such modification is phosphorylation, which happens to many enzymes and structural proteins in the process of cell signaling.TRANSCRIPT
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ABSTRACT METHODS RESULTS
FUTURE WORK
Brian Covello, Oliver Appelbe, Stephen Kron The University of Chicago, IL 60637 USA
APEX: A Novel Method for Proximity Proteomics within DNA Damage Induced Nuclear Foci and Subcellular Compartments
APEX Background ² APEX is an ascorbate peroxidase that was initially used as an EM tag in
the H2O2 dependent polymerization of diaminobenzidine (Rhee, 2013) ² Capable of oxidizing phenol derivatives such as biotin-tyramide,
ultimately causing a phenol radical reaction with Trp, Tyr, His, and Cys residues on nearby proteins (Rhee, 2013)
² Provides a novel mechanism for biotinylating proteins (Chapman-Smith, 1999)
² Fusion of APEX to proteins of interest allow insight into protein-protein interactions
Advantages " Reaction takes place in less than 1 millisecond, a short range for
biotinylation (Rhee, 2013) " Active in all cellular compartments, unlike horse radish peroxidase
(Howarth, 2008) " Allows for “proximity-dependent” proteomics, with a biotinylating
radius of approximately 20 nm (Rhee, 2013) " Allows for investigation of weak or transient interactions " Process can be regulated through H2O2 or biotin-tyramide concentration
Disadvantages " False negatives (low cellular protein concentration) " Specificity " Various histones bind streptavidin giving false positives (Roux, 2008)
Novel proteomic methods are providing enhanced insight into protein protein interactions. One such novel method is based off APEX, an ascorbate peroxidase responsible for catalyzing a reaction that leads to biotinylation of nearby proteins (Rhee, 2013). Unlike other conventional proteomic methods, APEX technology allows for investigation of insoluble proteins, weak or transient interactions, and non-direct interactors, as biotinylation of proteins occurs within a 20 nanometer radius (Rhee, 2013). This experiment sought to test the functionality and validity of APEX technology, to create an APEX template vector, and to fuse APEX to 53BP1 and RAD51 for the purpose of conducting proteomic analysis on these critical DNA damage response proteins. Validity of APEX was tested by targeting APEX to the mitochondria of MCF7 cells. Results indicate successful expression and functionality of mitochondria-APEX. Additionally, a novel gene construct, pTRIO-V5-APEX-53BP1 was engineered. This initial investigation provides a strong basis for conducting proteomic mapping of subcellular compartments and interactome analysis of the DNA damage response pathway.
CONCLUSIONS
ACKNOWLEDGEMENTS
ü Mito-APEX successfully expressed in MCF7 cells transfected with pcDNA3-mito-APEX
ü APEX expression unaffected by biotin-tyramide and hydrogen peroxide concentrations
ü 60 and 80 kDa biotinylated bands previously reported as evidence for APEX functionality were present in all lanes, control and experimental
ü Mito-APEX biotinylates proteins within mitochondria ü Biotinylation is up-regulated by increasing concentration of biotin-
tyramide ü pTRIO-V5-APEX template successfully engineered ü pTRIO-V5-APEX-53BP1 successfully engineered
» α-V5-HRP à APEX expression » No APEX expression in untransfected MCF7 cells, lanes 1-4 » APEX expression in all transfected MCF7 cells, lanes 5-10 » APEX expression is not affected by different combinations of biotin-
tyramide or H2O2
• Stephen Kron and Oliver Appelbe for mentorship and guidance • Andy Truman for cloning assistance • This project was funded by the National Science Foundation Molecular
Genetics Cellular Biology REU at the University of Chicago
APEX Technology & Reaction Conditions
Targeting APEX to the Mitochondria
① Transient transfection of MCF7 cells with construct of interest using FuGENE HD for 24 hours
② 30 minute incubation wi th 1x (500µM) biotin-tyramide
③ Catalyze reaction with 3 µ M H 2 O 2 f o r 1 minute
④ Quench reaction with antioxidants (trolox, sodium azide, sodium ascorbate)
REFERENCES
APEX Expression
Figure 11 (Previous research conducted by Rhee et al, 2013) » Initial success of mito-APEX was explained with streptavidin-HRP
staining of biotinylated protein bands at 60 and 80 kDa as indicated with arrows
Figure 12 (Results from our research) $ Biotinylated proteins at 60 and 80 kDa appearing in all lanes control and
experimental $ Significantly stronger staining with streptavidin-HRP occurs as the
concentration of biotin-tyramide is progressively increased throughout lanes 8-10, 2x = 1mM biotin-tyramide, 5x = 2.5 mM biotin-tyramide
$ Several protein bands appear in the experimental lanes 8-10 between 80-175 kDa and 50 kDa. These proteins bands are apparent only in lanes where mito-APEX is transfected and all the necessary components for the APEX reaction (biotin-tyramide and hydrogen peroxide) are present.
APEX-induced Biotinylation
q Mapping of subcellular proteins through mass spectrometry q Construction of pTRIO-V5-APEX-RAD51 q Experimentation with Constructs B in figure 7 as reported with
construct A q Stable expression of Constructs A and B in figure 7 through lentiviral
transfection q Proximity-dependent proteomic experiments using APEX technology in
pre-senescent and senescent cells q Enhancing APEX technology for utilization in cell-cycle specific phases q Elucidation of DNA damage response pathway through interactome
analysis
1. K. J. Roux, D. I. Kim, M. Raida, B. Burke, A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801 (2012). 2. M. Howarth, A. Y. Ting, Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat. Protoc. 3, 534 (2008). 3. A. Chapman-Smith, J. E. Cronan Jr., In vivo enzymatic protein biotinylation. Biomol. Eng. 16, 119 (1999). 4. H. Rhee, A.Y. Ting, Proteomic Mapping of Mitochondria in Living Cells via Spatially-Restricted Enzymatic Tagging. Science. 78, 1126 (2013). 5. I. Matsumura., Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques. 48, 6 (2010). 6. V. Tembe., Protein Trafficking in DNA Damage. Cell Signaling. 19, 2 (2007) 7. R.A. Greenberg., Histone Tails: Directing the chromatin response to DNA damage. FEBS Letters. 585, (2011).
Fig 1 Fig 2
Fig 3 Fig 4
Fig 5
Fig 6
Fig 10
Genetic Constructs
A. Validate functioning of APEX by demonstrating expression and biotinylation of APEX targeted to mitochondria
B. Create pTRIO-V5-APEX template for future use of creating fusion proteins
C. Fuse APEX to 53BP1 and RAD51 to conduct interactome analysis of proteins critical to the DNA damage response pathway
Ø V5 à Epitope Tag (α-V5-HRP detects APEX Expression) Ø Mito à Mitochondrial localization signal Ø Tet0Reg à Doxycycline inducible region
Ø 53BP1 à Truncated mRNA with IRIF foci signal Ø RAD51 à cDNA
Fig 13
(Rhee, 2013)
Fig 11 Fig 12
(Rhee, 2013)
DIRECTIVES
Picture courtesy of Tom Ellenberger at Washington University. Depicted is the initial stages of protein recruitment to a DNA double stranded break. Fusion of APEX to DNA repair proteins helps elucidate repair pathways.
Targeting APEX to DNA Damaged Foci
§ A = pcDNA3-‐mito-‐APEX
§ B = pTRIO-‐V5-‐APEX-‐53BP1
§ C = pTRIO-‐V5-‐APEX-‐RAD51
MCF7 (ATCC) MCF7 (53BP1) α-v5 mitotracker DAPI Merge α-v5 mitotracker DAPI Merge
Immunofluorescence Microscopy
→ Green = α-v5 with α-mouse AlexaFluor 488 (APEX expression) → Red = Mitotracker CMXRos with emission of 599 nm (Mitochondria) → Blue = DAPI with Emission of 461 nm (Nucleus) → Yellow = Overlap of APEX expression and mitochondria
Fig 9
Fig 7
Fig 8
(Tembe, 2007)
(Greenberg, 2011))