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Histone post-translational modifications in frontal cortex from human donors with Alzheimer's disease.

Anderson KW, Turko IV - Clin Proteomics (2015)

Bottom Line: Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases.Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability.Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioscience and Biotechnology Research, Rockville, MD 20850 USA ; Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 USA ; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 USA.

ABSTRACT

Background: Alzheimer's disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is partly due to the complexity of the disease and the largely unknown pathogenesis of sporadic AD, which accounts for the vast majority of cases. Epigenetics has been implicated as a critical component to AD pathology and a potential "hot spot" for treatments. Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases. Investigation of histone PTMs can help elucidate AD pathology and identify targets for therapies.

Results: A multiple reaction monitoring mass spectrometry assay was used to measure changes in abundance of several histone PTMs in frontal cortex from human donors affected with AD (n = 6) and age-matched, normal donors (n = 6). Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability. Additionally, a 91 % increase in ubiquitination of K120 on H2B was measured as well as an apparent loss in acetylation of the region near the N-terminus of H4. Our method of quantification was also determined to be precise and robust, signifying measured changes were representative of true biological differences between donors and sample groups.

Conclusion: We are the first to report changes in methylation of H2B K108, methylation of H4 R55, and ubiquitination of H2B K120 in frontal cortex from human donors with AD. These notable PTM changes may be of great importance in elucidating the epigenetic mechanism of AD as it relates to disease pathology. Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.

No MeSH data available.


Related in: MedlinePlus

Variability in replicate measurements. a The ratio of modified H3 peptide KacQLATKacAAR to H3 was measured in six replicate injections of a frontal cortex sample from a normal donor and individual ratios were normalized to their average (n = 6) and plotted to show the negligible variability between replicate injections. b The ratio of non-modified H2B to H3 plotted for biological replicates from normal donors (n = 6) and normalized to the average, showing negligible variability between biological replicates for the stoichiometric H2B/H3 ratio. c The ratio of KacQLATKacAAR to H3 plotted for the average of two injections for each biological sample from normal donors (n = 6) and normalized to the average for all measurements (n = 12), showing variability in PTM is attributed to differences in donors and not due to inherent variability in MRM
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Fig1: Variability in replicate measurements. a The ratio of modified H3 peptide KacQLATKacAAR to H3 was measured in six replicate injections of a frontal cortex sample from a normal donor and individual ratios were normalized to their average (n = 6) and plotted to show the negligible variability between replicate injections. b The ratio of non-modified H2B to H3 plotted for biological replicates from normal donors (n = 6) and normalized to the average, showing negligible variability between biological replicates for the stoichiometric H2B/H3 ratio. c The ratio of KacQLATKacAAR to H3 plotted for the average of two injections for each biological sample from normal donors (n = 6) and normalized to the average for all measurements (n = 12), showing variability in PTM is attributed to differences in donors and not due to inherent variability in MRM

Mentions: Selected transitions for quantification had Gaussian peak shape with more than ten data points across each peak and suitable intensity as is preferred for quantification (Additional file 3: Figure S1). However, it is important to establish the level of variability in measurements to confirm that observed changes are attributed to true biological changes and not an artifact of LC–MS/MS performance or sample preparation. To demonstrate our data quality, we first performed six replicate injections of the same sample (Fig. 1a) and measured modified H3 peptide KacQLATKacAAR as a ratio to non-modified H3 peptide DIQLAR. Measured ratios were nearly identical with negligible variation (Fig. 1a), demonstrating LC–MS/MS performance was precise and not a contributor to measurement variation. Second, we compared variability between six biological samples from normal frontal cortex using the ratio of non-modified H2B peptide EIQTAVR to H3 peptide DIQLAR (Fig. 1b). Histones H2B and H3 are present in equal amounts as there is a 1:1 stoichiometry between core histones in the nucleosome. Therefore, H2B to H3 should not change between donors or sample preparations. Indeed, our measurements confirm that there is negligible variation in H2B/H3 between donors, demonstrating that our sample preparation results in precision of measurements. Lastly, we monitored the H3 peptide KacQLATKacAAR as a ratio to H3 peptide DIQLAR (Fig. 1c) for the same six donors of normal frontal cortex and in the same LC–MS/MS runs as for Fig. 1b. Detectable variability across donors for KacQLATKacAAR shows that variability in KacQLATKacAAR measurements is due to biological differences in PTM level between donors. In summary, we established that measured differences between AD and control samples described herein are representative of biological variation and that our sample preparation and MRM assay is robust and precise.Fig. 1


Histone post-translational modifications in frontal cortex from human donors with Alzheimer's disease.

Anderson KW, Turko IV - Clin Proteomics (2015)

Variability in replicate measurements. a The ratio of modified H3 peptide KacQLATKacAAR to H3 was measured in six replicate injections of a frontal cortex sample from a normal donor and individual ratios were normalized to their average (n = 6) and plotted to show the negligible variability between replicate injections. b The ratio of non-modified H2B to H3 plotted for biological replicates from normal donors (n = 6) and normalized to the average, showing negligible variability between biological replicates for the stoichiometric H2B/H3 ratio. c The ratio of KacQLATKacAAR to H3 plotted for the average of two injections for each biological sample from normal donors (n = 6) and normalized to the average for all measurements (n = 12), showing variability in PTM is attributed to differences in donors and not due to inherent variability in MRM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4591557&req=5

Fig1: Variability in replicate measurements. a The ratio of modified H3 peptide KacQLATKacAAR to H3 was measured in six replicate injections of a frontal cortex sample from a normal donor and individual ratios were normalized to their average (n = 6) and plotted to show the negligible variability between replicate injections. b The ratio of non-modified H2B to H3 plotted for biological replicates from normal donors (n = 6) and normalized to the average, showing negligible variability between biological replicates for the stoichiometric H2B/H3 ratio. c The ratio of KacQLATKacAAR to H3 plotted for the average of two injections for each biological sample from normal donors (n = 6) and normalized to the average for all measurements (n = 12), showing variability in PTM is attributed to differences in donors and not due to inherent variability in MRM
Mentions: Selected transitions for quantification had Gaussian peak shape with more than ten data points across each peak and suitable intensity as is preferred for quantification (Additional file 3: Figure S1). However, it is important to establish the level of variability in measurements to confirm that observed changes are attributed to true biological changes and not an artifact of LC–MS/MS performance or sample preparation. To demonstrate our data quality, we first performed six replicate injections of the same sample (Fig. 1a) and measured modified H3 peptide KacQLATKacAAR as a ratio to non-modified H3 peptide DIQLAR. Measured ratios were nearly identical with negligible variation (Fig. 1a), demonstrating LC–MS/MS performance was precise and not a contributor to measurement variation. Second, we compared variability between six biological samples from normal frontal cortex using the ratio of non-modified H2B peptide EIQTAVR to H3 peptide DIQLAR (Fig. 1b). Histones H2B and H3 are present in equal amounts as there is a 1:1 stoichiometry between core histones in the nucleosome. Therefore, H2B to H3 should not change between donors or sample preparations. Indeed, our measurements confirm that there is negligible variation in H2B/H3 between donors, demonstrating that our sample preparation results in precision of measurements. Lastly, we monitored the H3 peptide KacQLATKacAAR as a ratio to H3 peptide DIQLAR (Fig. 1c) for the same six donors of normal frontal cortex and in the same LC–MS/MS runs as for Fig. 1b. Detectable variability across donors for KacQLATKacAAR shows that variability in KacQLATKacAAR measurements is due to biological differences in PTM level between donors. In summary, we established that measured differences between AD and control samples described herein are representative of biological variation and that our sample preparation and MRM assay is robust and precise.Fig. 1

Bottom Line: Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases.Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability.Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bioscience and Biotechnology Research, Rockville, MD 20850 USA ; Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 USA ; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 USA.

ABSTRACT

Background: Alzheimer's disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is partly due to the complexity of the disease and the largely unknown pathogenesis of sporadic AD, which accounts for the vast majority of cases. Epigenetics has been implicated as a critical component to AD pathology and a potential "hot spot" for treatments. Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases. Investigation of histone PTMs can help elucidate AD pathology and identify targets for therapies.

Results: A multiple reaction monitoring mass spectrometry assay was used to measure changes in abundance of several histone PTMs in frontal cortex from human donors affected with AD (n = 6) and age-matched, normal donors (n = 6). Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability. Additionally, a 91 % increase in ubiquitination of K120 on H2B was measured as well as an apparent loss in acetylation of the region near the N-terminus of H4. Our method of quantification was also determined to be precise and robust, signifying measured changes were representative of true biological differences between donors and sample groups.

Conclusion: We are the first to report changes in methylation of H2B K108, methylation of H4 R55, and ubiquitination of H2B K120 in frontal cortex from human donors with AD. These notable PTM changes may be of great importance in elucidating the epigenetic mechanism of AD as it relates to disease pathology. Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.

No MeSH data available.


Related in: MedlinePlus