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![Page 1: Thesis Presentation](https://reader036.fdocumento.com/reader036/viewer/2022081604/587a95131a28ab09758b49ef/html5/thumbnails/1.jpg)
Measuring mitochondrial
nonenzymatic lysine acetylation
Keighley ReisenauerDr. John Denu & Josue BaezaUW – Madison Department of Biomolecular ChemistryWisconsin Institute of Discovery | Epigenetics Theme
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What is acetylation?
Acetylation’s role in mitochondria
Experiment process
Data analysis
Future directions
Outline
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denulab.discovery.wisc.edu
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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
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What are the roles of acetylation?
“What's Up and Down with Histone Deacetylation and Transcription?” Pazin, Michael J, James T Kadonaga: Cell, 1997
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Model of acetylation in mitochondria
http://www.lab.hirschey.org/resources/Essays-Biochem.-2012-Anderson.pdf
low acetylationhigh acetylation
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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
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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
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What is the protein purification workflow?
E. Coli flask, Josh Baezahttp://www.wired.com/2014/05/synthetic-dna-cells/
Waste
FPLC
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What does an FPLC trace determine?
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Assay workflow
“Site-Specific Reactivity of Nonenzymatic Lysine Acetylation,” Josue Baeza, Michael Smallegan, John Denu. ACS Chemical Biology, 2015
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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
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Previous data
“Site-Specific Reactivity of Nonenzymatic Lysine Acetylation,” Josue Baeza, Michael Smallegan, John Denu. ACS Chemical Biology, 2015
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What are the mass spec data results?
Protein Site Reactivity
Acetyl-R
PHD K244 0.9589 Ac-CoACKMT K128 0.9879 Ac-CoA
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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
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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
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ACKNOWLEDGEMENTSThank you to Dr. John Denu and Josue Baeza for all their
support
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QUESTIONS?