![Keighley Reisenauer May 1, 2016
John Denu
Biomolecular Chemistry
REFERENCES
1. Wagner, Gregory R, Matthew D. Hirschey (2014). Nonenzymatic Protein Acylation as a
Carbon Stress Regulated by Sirtuin Deacylases. Molecular Cell 54, 5-16.
2. National Center for Biotechnology Information RefSeq (2008). SIRT3 sirtuin 3 [Homo
sapiens (human)]. < http://www.ncbi.nlm.nih.gov/gene/23410>
3. Pazin, Michael J, James T Kadonaga (1997). What's Up and Down with Histone
Deacetylation and Transcription? Cell 89, 325–328.
4. Sadoul, Karin, et. al. (2008).Regulation of protein turnover by acetyltransferases and
deacetylases. Biochimie 90, 306–312.
5. Zhao, Shimin, et. al. (2010). Regulation of Cellular Metabolism by Protein Lysine
Acetylation. Science 19, 1000-1004.
6. Osborne, Brenna, Gregory J. Cooneya, Nigel Turner. Are sirtuin deacylase enzymes
important modulators of mitochondrial energy metabolism? (2014). Biochimica et
Biophysica Acta (BBA) - General Subjects 1840:4, 295–1302.
7. Baeza, Josue, Michael J Smallegan, and John M Denu. Mechanisms and Dynamics of
Protein Acetylation in Mitochondria. (2016). Cell Press, 231-44.
8. Sack, Michael N. The role of SIRT3 in mitochondrial homeostasis and cardiac adaptation
to hypertrophy and aging. (2012). Journal of Molecular and Cellular Cardiology 53: 3,
520-525.
9. Weinert, Brian T, et. al. Analysis of acetylation stoichiometry suggests that SIRT3 repairs
nonenzymatic acetylation lesions. (2015). The Embo Journal 34, 2620-32.
10. Hirschey MD, et. al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible
enzyme deacetylation. (2010). Nature 464, 121–125.
11. Glozak, M.A., N. Sengupta, X. Zhang, E. Seto. Acetylation and deacetylation of non-
histone proteins. (2005). Gene 363:1-2, 15-23.
12. Allfrey, V.G., R. Faulkner, A.E. Mirsky. Acetylation and methylation of histones and
their possible role in the regulation of RNA synthesis. (1964). Biochemistry 51, 786-94
13. Pougovkina O, et. al. Mitochondrial protein acetylation is driven by acetyl-CoA from
fatty acid oxidation. (2014). Human Molecular Genetics 23, 3513–22.
14. Baeza, Josue, Michael J Smallegan, and John M Denu. Site-Specific Reactivity of
Nonenzymatic Lysine Acetylation. (2015). ACS Chemical Biology 10. 122-8.
15. Brock, Tom. Protein Acetylation: Much More than Histone Acetylation. (2015).
Epigenetics, Cayman Chemical.
16. Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A. Histone acetylation and
disease. (2001). Cell Molecular Life Science, Volume 58. 728-736.
17. GR Wagner, MR Payne. Mitochondrial Acetylation and Diseases of Aging. (2011).
Journal of Aging Research, Volume 2011.](https://image.slidesharecdn.com/4339ef29-9309-46de-bb1e-d5d088893a75-160720203542/85/20160501_Final-9-320.jpg)
20160501_Final
- 1. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry MEASURING MITOCHONDRIAL NONENZYMATIC LYSINE ACETYLATION Abstract Protein acetylation is a regulatory modification affecting numerous biochemical and cellular processes. Over half of the proteins in mitochondria have been identified as acetylated, however the mechanism of how this occurs has not been elucidated. This project focuses on understanding the mechanism of mitochondrial protein acetylation, hypothesizing that non- enzymatic acetylation is responsible for the majority of observed acetylation. In order to understand non-enzymatic acetylation, we must be able to quantify this reaction. Quantifying nonenzymatic acetylation is accomplished by incubating a protein with increasing concentrations of acetyl-CoA. The rate is determined by plotting the change in acetylation over concentration of acetyl-CoA. The slope of the line provides the second order rate constant for individual lysine reactivity in native proteins. This study will focus on the acetylation mechanism for the following mitochondrial proteins: ACAT, PDH, HMGCS2, HMGCL1, αKGDH, and CKMT, which are reported in the scientific literature to be highly acetylated in vivo. Introduction Mitochondria are highly dynamic organelles that exist within a complex metabolic network to allow for cell-wide adjustments to stresses and energy demands (Osborne, 2014). The critical role of mitochondria in regulating this cellular homeostasis is highlighted by defects in mitochondrial function such as impaired oxidative phosphorylation, excessive reactive oxygen species (ROS), and altered mitochondrial dynamics. These defects can result a wide range of disease including aging diseases, cancer, fragile X, or heart failure (Brock, 2010; Timmermann, 2001; Wager, 2011). Some of these defects can arise from a downstream effect of post-translational
- 2. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry modifications (PTMs) that lead to impaired protein function within the cell (Osborne, 2014; Sack, 2012). One type of PTM is acetylation, which has been shown to affect protein turnover and intermediate catabolic metabolism (Zhao, 2010; Sadoul, 2008). Acetylation is the process by which an acetyl group is added to an organic macromolecule, such as a protein. When a lysine is acetylated, the acetyl group neutralizes the lysine’s positive charge, thus decreasing affinity for negatively charged molecules, which can impair protein structure and stability. This defective protein could then have impaired enzymatic activity, interactions, and localization of target proteins (Glozak, 2005). Several possible functions of acetylation were proposed in Figure 1 (Pazin and Kadonaga, 1997). Because acetylation can occur at many residues, there is no singular effect. The effect of acetylation is dependent on the site of acetylation on the protein and can result in protein conformational changes, increases or decreases in signaling, or protein activity changes. Historically, acetylation of nuclear histone proteins became the first well-established example of functional protein acetylation when it was discovered more than fifty years ago (Allfrey, 1964). It wasn’t until the late 1990s that the first histone acetyltransferases (HATs) and deacetylases (HDACs) were cloned and linked to the regulation of gene expression and chromatin structure (Baeza, 2016; Osborne, 2014). Acetylation acts in part to “open up” chromatin for appropriate transcriptional machinery to access the DNA template (Baeza, 2016). Advances in technology like innovative immunoprecipitation techniques and the development of liquid chromatography coupled mass spectrometry (LC-MS) provided lists of acetylated peptides with their corresponding proteins (Baeza, 2016). Subsequent observations of extra-nuclear deacetylase localization and notable enrichment of mitochondrial metabolic proteins spurred a greater interest in exploring the acetyl-proteome outside the nucleus (Verdin and Ott, 2016).
- 3. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Studies have found that 63% of mitochondrially localized proteins contain acetylation sites, both in mouse and human models (Baeza, 2016). Unlike nuclear acetylation, which is catalyzed by several families of lysine acetyltransferases (KATs), mitochondrial acetylation is considered to be largely nonenzymatic. This hypothesis is driven by the lack of direct evidence of KATs in mitochondria, particularly in the high pH environment of the mitochondrial matrix (Baeza, 2015; Osborne, 2014; Pougovkina, 2014; Wagner and Hirschey, 2014). Generally in the mitochondria, protein acetylation typically leads to loss of function in pathways associated with organelle integrity and oxidative metabolism (Baeza, 2016; Weinert, 2015). Lysine acetylation, specifically, has been shown to affect protein turnover and intermediate catabolic metabolism (Zhao, 2010; Sadoul, 2008). A study by Baeza, et. al. reveals that oxidative metabolism is inhibited by higher levels of acetylation among certain metabolic enzymes (Table 1, Baeza, 2016). The study continued to evaluate these enzymes’ morphology and observed that lysine sites with the highest acetylation reactivity tend to protrude from the protein surface, while low reactivity sites are protected by electrostatic interactions with neighboring residues (Figure 2, Baeza, 2016). Both surface exposure and local electrostatic interactions influence lysine reactivity toward acetyl- phosphate and acetyl-CoA, but computed pKa values were not a reliable predictor of lysine reactivity (Baeza, 2016). Sirtuin 3 (SIRT3) acts along these proteins in the mitochondria, but with deacetylation activity (Onyango, 2002). In yeast, “sirtuin proteins are known to regulate epigenetic gene silencing and suppress recombination of rDNA” (NCIB RefSeq 2008). However, unlike typical deacetylases, sirtuins acetylate proteins by consuming NAD+ and releasing nicotinamide, O-acetyl ADP ribose (OAADPr), and the deacetylated substrate (Fig. 3; Osborne, 2014). Overall, SIRT3 modulates mitochondrial homeostasis and targets proteins involved in mediating energy
- 4. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry metabolism and mitochondrial redox stress (Sack, 2012). In mitochondria, highly reactive sites that exist in clusters of lysine resides are high-affinity substrates of SIRT3 activity, which can lead to enhanced oxidative metabolism by reversing the inhibitory effect of acetylation (Baeza, 2016). Table 1 lists central enzymes that have reversed acetylation by SIRT3 and a rescued function. SIRT3 expression has been shown to increase by fasting and chronic caloric restriction, two conditions that necessitate increased oxidative metabolism (Baeza, 2016; Hirschey, 2010; Sack, 2016; Weinert, 2015). It is not surprising, then, that SIRT3 is involved in the deacetylation of enzymatic proteins that facilitate the conversion of acetate to acetyl-CoA for energy production (Sack, 2012). SIRT3 works to combat and regulate acetylation “carbon stress/ buildup” to ensure metabolic fidelity (Wagner and Hirschey, 2014). Regulated by SIRT3, steady-state mitochondrial acetylation only presents at very low levels, even in response to caloric restriction, suggesting that highly acetylated mitochondria are rapidly turned over to preserve organelle-wide functionality (Baeza, 2016; Weinert, 2015). In contrast, a SIRT3 knock-out results in substantially increased acetylation at target sites that is significantly increased from any changes observed at these same sites in fasted or calorie-restricted conditions (Weinert, 2015). This indicates that SIRT3 has a greater role in acetylation suppression than the effects of just dietary manipulations. Further effects of sirtuin knock-out are an increase of ROS and an alteration of oxidative metabolism, suggesting SIRT3 plays a major role in fine-tuning multiple metabolic programs in response to caloric and redox stressors (Baeza, 2016; Sack, 2012). The similarities between nuclear and mitochondrial protein (de)acetylation is striking and leads to a need for innovative techniques to study the differences. While nuclear acetylation is enzyme-catalyzed and results in upregulated processes, mitochondrial nonenzymatic acetylation typically results in a loss of function, particularly in proteins involved in oxidative metabolism
- 5. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry (Baeza, 2016). However, a more complete picture of the role of acetylation in mitochondria is required and stoichiometric approaches provide new methods to analyzing mitochondrial lysine acetylation and evaluating the nonenzymatic activity. Stoichiometry is a means to evaluate the fraction of a protein that is modified, instead of measuring fold-change. Figure 4 provides a visual representation between the differences between fold-change and stoichiometric analysis. Stoichiometry can discern low-level changes in acetylation more accurately, as well as focus on site-specific changes. Across multiple sites, low-level stoichiometry can have additive effects on protein activity. Acetyl sites on the same molecule or acetyl sites on different molecules that share similar function could collectively dampen or enhance protein activity, in accordance with how many sites are occupied (Baeza, 2015). Following the schematic in Figure 5, this study aims to employ stoichiometric analysis to quantify the acetylation reactions of ACAT, PDH, HMGCS2, HMGCL1, and αKGDH mitochondrial proteins. Due to the evidence surrounding the mitochondrial environment and lack of lysine acetyltransferase enzymes, it is hypothesized that majority of mitochondrial lysine acetylation will be result from nonenzymatic reactions, regulated to low stoichiometric levels by SIRT3. Site specific stoichiometry is determined by reacting acetyl- CoA or acetyl-phosphate with native proteins, denaturing the product, and reacting with heavy- labeled acetic anhydride to chemically acetylate all remaining unmodified lysines. The rate is determined by plotting the change in acetylation over concentration of acetyl-CoA. The slope of the line provides the second order rate constant for individual lysine reactivity in native proteins. It is through these approaches and the support from previous literature that this study will focus on the acetylation mechanism for the following mitochondrial proteins: ACAT, PDH, HMGCS2, HMGCL1, αKGDH, and CKMT.
- 6. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Methods Purification of Recombinant proteins E. coli cells were transfected and grown up in liter cultures to express the protein of interest. Then, the cells were lysed and the protein was purified using fast protein liquid chromatography (FPLC) with nickel affinity resin. (Figure 6). SDS-PAGE gels confirmed purity of the protein. Concentration of protein was then determined via Bradford Assay. Chemical Acetylation Kinetics The purified protein was treated with increasing concentrations (0.5 mM, 1 mM, 2mM, 4 mM, and 8mM) of either acetyl-CoA or acetyl-P, causing acetylation at particular lysine residues. Incubation for one hour with an isotopic acetyl group labeled the remaining unmodified residues in vitro (Figure 7). After treatment, the pH was restored to pH 8 using ammonium hydroxide to hydrolyze any O-acetyl esters formed during the reaction. Then, the labeled proteins were spin filtered and digested using trypsin at a 1:100 ratio overnight. Mass Spectrometry The sample was cleaned using Stage Tips with C18 carbon activated with methanol. This allows the filter to become “wet” and is used preferentially to water, which would not be able to soak the organic filter material. The filter was then washed with 80% ACN, a nonpolar solvent, which is at the same concentration as the elution solution. A secondary wash with 0.5% acetic acid effectively replaced the CAN and stimulate peptide binding. The sample was then loaded and again washed with 0.5% acetic acid to remove any salts, urea, or small molecules. The acetylated peptides were then stripped from the filter using 80% ACN, which disrupts the peptide’s ability to bind. This elution was then dried to be ready for mass spectrometry. After this protein digestion and
- 7. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry desalting, the peptides were injected into the mass spectrometer and the relative abundance of the light and heavy acetyl peptides were measured. Results / Discussion The data from the mass spectrometer was analyzed for two proteins. We ran into delays with the remaining proteins due to malfunctions with the MS machine and did not have time to run and analyze the other four purified proteins. We used the Mascot database search engine to determine the lysine residue locations on each protein. We successfully identified one lysine site on pyruvate dehydrogenase (PDH) and the reactivity was significantly higher with Acetyl-CoA than with Acetyl-P (rate = 1.0561 1*105 M-1s-1 and rate = 0.0083 1*105 M-1s-1, respectively). We identified four lysine sites on mitochondrial creatine kinase (CKMT) and all sites were consistently more reactive with Acetyl-CoA compared to Acetyl-P. The most reactive lysine was at K313, 321 (Figure 8). The graphs also suggest that there is a linearly increasing correlation between the acetyl-R concentration and the protein acetylation. Conclusions / Future Directions The next steps for data analysis include enzyme activity assays and protein modeling. The assays will help determine the protein’s acetylation tolerance as we increase acetyl-R concentration. Each enzyme will be incubated with either Acetyl-CoA or Acetyl-Phosphate using conditions outlined above. The enzyme activity will then be assessed by measuring the increase in absorbance of product formation using steady-state conditions. Secondly, oxidative metabolism is inhibited by higher levels of acetylation among certain metabolic enzymes (Baeza, 2016). Baeza et. al. evaluated the second order rate constant with the crystal structure of metabolic enzymes and observed that lysine sites with the highest acetylation reactivity tend to protrude from the protein
- 8. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry surface, while low reactivity sites are protected by electrostatic interactions with neighboring residues (Figure 9). We intend to complete similar structures for the remaining proteins in the study to capture a three-dimensional and more comprehensive view of the protein’s acetylations. Ideally, we will expand our work to include other mitochondrial proteins to more fully understand the acetylation process. Acknowledgements I would like to thank Dr. John Denu for the opportunity to complete this research under his guidance and the support of Josue Baeza. I would also like to thank the CALS Honors Program for this opportunity and the privilege of earning Honors distinctions.
- 9. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry REFERENCES 1. Wagner, Gregory R, Matthew D. Hirschey (2014). Nonenzymatic Protein Acylation as a Carbon Stress Regulated by Sirtuin Deacylases. Molecular Cell 54, 5-16. 2. National Center for Biotechnology Information RefSeq (2008). SIRT3 sirtuin 3 [Homo sapiens (human)]. < http://www.ncbi.nlm.nih.gov/gene/23410> 3. Pazin, Michael J, James T Kadonaga (1997). What's Up and Down with Histone Deacetylation and Transcription? Cell 89, 325–328. 4. Sadoul, Karin, et. al. (2008).Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie 90, 306–312. 5. Zhao, Shimin, et. al. (2010). Regulation of Cellular Metabolism by Protein Lysine Acetylation. Science 19, 1000-1004. 6. Osborne, Brenna, Gregory J. Cooneya, Nigel Turner. Are sirtuin deacylase enzymes important modulators of mitochondrial energy metabolism? (2014). Biochimica et Biophysica Acta (BBA) - General Subjects 1840:4, 295–1302. 7. Baeza, Josue, Michael J Smallegan, and John M Denu. Mechanisms and Dynamics of Protein Acetylation in Mitochondria. (2016). Cell Press, 231-44. 8. Sack, Michael N. The role of SIRT3 in mitochondrial homeostasis and cardiac adaptation to hypertrophy and aging. (2012). Journal of Molecular and Cellular Cardiology 53: 3, 520-525. 9. Weinert, Brian T, et. al. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions. (2015). The Embo Journal 34, 2620-32. 10. Hirschey MD, et. al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. (2010). Nature 464, 121–125. 11. Glozak, M.A., N. Sengupta, X. Zhang, E. Seto. Acetylation and deacetylation of non- histone proteins. (2005). Gene 363:1-2, 15-23. 12. Allfrey, V.G., R. Faulkner, A.E. Mirsky. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. (1964). Biochemistry 51, 786-94 13. Pougovkina O, et. al. Mitochondrial protein acetylation is driven by acetyl-CoA from fatty acid oxidation. (2014). Human Molecular Genetics 23, 3513–22. 14. Baeza, Josue, Michael J Smallegan, and John M Denu. Site-Specific Reactivity of Nonenzymatic Lysine Acetylation. (2015). ACS Chemical Biology 10. 122-8. 15. Brock, Tom. Protein Acetylation: Much More than Histone Acetylation. (2015). Epigenetics, Cayman Chemical. 16. Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A. Histone acetylation and disease. (2001). Cell Molecular Life Science, Volume 58. 728-736. 17. GR Wagner, MR Payne. Mitochondrial Acetylation and Diseases of Aging. (2011). Journal of Aging Research, Volume 2011.
- 10. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry 18. Verdin E. and Ott M. 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. (2015). Nature Review Molecular Cell Biology 4, 258-64.
- 11. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry APPENDIX Figure 1. Examples of possible mechanisms for protein acetylation. Taken fromPazin and Kadonaga, 1997. Table 1. List of identified proteins associated with central metabolismthat are acetylated.Taken fromBaeza, et. al, 2015
- 12. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Figure 2. Diagramof lysine reactivity. Red is high reactivity and yellow is low reactivity. Lysine residues that stick out or are near the acetyl-CoA binding pocket tend to react more strongly than those that are bound internally and are protected by electrostatic interactions with nearby molecules. Taken from Baeza,et. al. 2015. Figure 3. Diagram of acetyl-CoA consumption pathways. Included is a schematic depicting acetylation and deacetylation of proteins using acetyl-CoA. Taken from Osborne 2014.
- 13. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Figure 4. Diagram describing the difference between fold-change and stoichiometric analysis of protein acetylation. Low-level changes are able to be captured with stoichiometric approaches. Taken from Josh Baeza. Figure 5. Kinetic approachesof nonenzymatic lysine acetylation.A. Diagramof methodology used to determine nonenzymatic acetylation. Purified mitochondrial and non-mitochondrial proteins assayed with varying concentrations of acetyl-phosphate or acetyl-CoA. B. Time dependent acetylation of bovine serumalbumin showing a linear increase of site-specific lysine acetylation. C. Concentration dependent acetylation of glutamate dehydrogenase, showing a linear increase of rate constants. D. Dot plot of rate constants analyzed in the study. Taken from Baeza, et. al., 2015
- 14. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Figure 6. Visual representation of recombinant protein purification. The E. coli cells are grown in 1L of media,then the protein (represented in pink) is extracted and run on the FPLC over a nickel-affinity column to purify the tagged protein of choice. A spectrometer determines the peak output of the desired protein, which can be analyzed and run on a SDS-PAGE gel for size consistency. This increasesconfidence that the resulting protein is pure. Figure 7. Visual representation of chemical acetylation kinetics.The diagramdepicts the lysine (K) residues on the purified protein, then the incubation with the acetyl-R group in increasing concentrations. Some of the lysine restudies are then acetylated, and further processing labels the remaining lysine residues with a heavy acetyl-R group. Next stepsinvolve running the processed protein on the mass spectrometer and further data analysis. Taken from Baeza, Smallegan, & Denu, 2015.
- 15. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Figure 8. In the graphs above, the blue points represent concentrations of acetyl-Co vs acetylation. The red points, acetyl-phosphate (Ac-P). The table describes the reactivity of a specific lysine site on the proteins from mass spectrometry analysis. All sites react more with Ac-CoA than with Ac-P. The graphs also suggest that the relationship between acetyl-R concentration and protein acetylation is linearly increasing.
- 16. Keighley Reisenauer May 1, 2016 John Denu Biomolecular Chemistry Figure 9. Diagram of lysine reactivity. Red is high reactivity and yellow is low reactivity. Lysine residues that stick out or are near the acetyl-CoA binding pocket tend to react more strongly than those that are bound internally and are protected by electrostatic interactions with nearby molecules. Taken from Baeza, Smallegan, & Denu, 2015.