Measuring mitochondrial

nonenzymatic lysine acetylation

Keighley ReisenauerDr. John Denu & Josue BaezaUW – Madison Department of Biomolecular ChemistryWisconsin Institute of Discovery | Epigenetics Theme

What is acetylation?

Acetylation’s role in mitochondria

Experiment process

Data analysis

Future directions

Outline

denulab.discovery.wisc.edu

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

What are the roles of acetylation?

“What's Up and Down with Histone Deacetylation and Transcription?” Pazin, Michael J, James T Kadonaga: Cell, 1997

Model of acetylation in mitochondria

http://www.lab.hirschey.org/resources/Essays-Biochem.-2012-Anderson.pdf

low acetylationhigh acetylation

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

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

What is the protein purification workflow?

E. Coli flask, Josh Baezahttp://www.wired.com/2014/05/synthetic-dna-cells/

Waste

FPLC

What does an FPLC trace determine?

Assay workflow

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

What are the proteins currently studied?

Ketogenesis, Josh Baezahttps://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/krebs.htm

ACATHMGCSHMGCL

PDHaKGDHCKMT

ketogenesis

TCA cyclecreatine

metabolism

Previous data

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

What are the mass spec data results?

Protein Site Reactivity

Acetyl-R

PHD K244 0.9589 Ac-CoACKMT K128 0.9879 Ac-CoA

Visualizing rate constants

Second order rate constant1. Physical location 2. Proximity to Acetyl Co-A pockets3. 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

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

ACKNOWLEDGEMENTSThank you to Dr. John Denu and Josue Baeza for all their

support

QUESTIONS?