1. Measuring mitochondrial
nonenzymatic lysine
acetylation
Keighley Reisenauer
Dr. John Denu & Josue Baeza
UW – Madison
Department of Biomolecular Chemistry
Wisconsin Institute of Discovery | Epigenetics Theme

2. What is acetylation?
Acetylation’s role in mitochondria
Experiment process
Data analysis
Future directions
Outline

3. denulab.discovery.wisc.edu

4. Why is acetylation important?
Acetylation
Deacetylation
 Gene expression
regulation
 Protein stability
 Aging
 Disease
 Leukemia
 Fragile X
 Heart failure
“Protein Acetylation: Much More than Histone Acetylation,” Tom Brock: Epigenetics, 2010
“Histone acetylation and disease,” Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A: Cell Molecular Life Science, 2001
“Mitochondrial Acetylation and Diseases of Aging,“ GR Wagner, MR Payne: Journal of Aging Research, 2011

5. What are the roles of acetylation?
“What's Up and Down with Histone Deacetylation and Transcription?” Pazin, Michael J, James T Kadonaga: Cell, 1997

6. Model of acetylation in mitochondria
http://www.lab.hirschey.org/resources/Essays-Biochem.-2012-Anderson.pdf

7. Hypothesis
Nonenzymatic acetylation is influenced by
the reactivity of lysine residues, which is
affected by the local environment.
Big picture: understand the mechanism of
acetylation

8. In-vitro kinetic data analysis
Mass spec analysis
Mass spec sample prep
Protein incubation with acetyl-group gradient in vitro
Protein Purification (FPLC)
Grow E. coli cells with protein
Methods workflow

9. What is the protein purification workflow?
E. Coli flask, Josh Baeza
http://www.wired.com/2014/05/synthetic-dna-cells/
Waste
FPLC

10. What does an FPLC trace determine?

11. Assay workflow
“Site-Specific Reactivity of Nonenzymatic Lysine Acetylation,” Josue
Baeza, Michael Smallegan, John Denu. ACS Chemical Biology, 2015

12. What are the proteins currently studied?
Ketogenesis, Josh Baeza
https://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/krebs.htm
ACAT
HMGCS
HMGCL
PDH
aKGDH
CKMT

13. Previous data
“Site-Specific Reactivity of Nonenzymatic Lysine Acetylation,” Josue
Baeza, Michael Smallegan, John Denu. ACS Chemical Biology, 2015

14. What are the mass spec data results?
Protein Site Reactivity Acetyl-R
PHD K244 0.9589 Ac-CoA
CKMT K128 0.9879 Ac-CoA

15. Visualizing rate constants
 Second order rate constant
1. Physical location
2. Proximity to Acetyl Co-A pockets
3. Nearby chemical interactions
4. Other amino acids in the vicinity
“Site-Specific Reactivity of Nonenzymatic Lysine Acetylation,” Josue
Baeza, Michael Smallegan, John Denu. ACS Chemical Biology, 2015

16. Overarching goal: understanding the mechanisms of
acetylation (implicated in aging, diseases, etc.)
 Continue to investigate mass spectrometry data of studied
proteins
 Include more proteins in the study; reach out to other
groups for collaboration
 It is shown that increased activity of Sirtuins is linked to
increased lifespan; by investigating what drives
acetylation, we can better understand the aging process
Summary and Future Directions

17. ACKNOWLEDGEMENTS
Thank you to Dr. John Denu and Josue Baeza for all their support

18. QUESTIONS?