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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

Histone PTMs in frontal cortex. a Measured peak areas of each quantified peptide were normalized to the peak area of different histone subunits. The relative standard deviation (n = 12) for each quantified peptide in normal and AD frontal cortex is shown grouped by the histone in which the peptides are located. The identity of the PTM measurements is presented in the graph in b, which appears in the same order. b Changes in histone PTMs in AD-affected human frontal cortex. Measurements were performed using human frontal cortex from normal (n = 6) and AD-affected (n = 6) donors (Additional file 1: Table S1) and two experimental replicates per donor (n = 12 total measurements per condition). Q-peptide transitions are summarized in Table 1. Data presented as mean ± SD. PTMs are (ac) acetylation, (me) methylation, and (ub) ubquitination. *p < 0.05; **p < 0.01; ***p < 0.001
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Fig2: Histone PTMs in frontal cortex. a Measured peak areas of each quantified peptide were normalized to the peak area of different histone subunits. The relative standard deviation (n = 12) for each quantified peptide in normal and AD frontal cortex is shown grouped by the histone in which the peptides are located. The identity of the PTM measurements is presented in the graph in b, which appears in the same order. b Changes in histone PTMs in AD-affected human frontal cortex. Measurements were performed using human frontal cortex from normal (n = 6) and AD-affected (n = 6) donors (Additional file 1: Table S1) and two experimental replicates per donor (n = 12 total measurements per condition). Q-peptide transitions are summarized in Table 1. Data presented as mean ± SD. PTMs are (ac) acetylation, (me) methylation, and (ub) ubquitination. *p < 0.05; **p < 0.01; ***p < 0.001

Mentions: Changes in quantified histone PTMs between AD and normal frontal cortex from human donors are presented in Fig. 2b. For quantification of histone PTMs, the peak areas of modified peptides were normalized to the peak areas of non-modified peptides from the respective histone. For example, ubiquitinated H2B peptide AVTKubYTSSK was normalized to H2B peptide EIQTAVR while acetylated H4 peptide GLGKacGGAKacR was normalized to H4 peptide VFLENVIR. While normalization of PTMs within a protein to the total amount of the protein is an accepted practice for quantification [20], it is typically not advisable to normalize PTMs within one protein to the total abundance of a different protein. Histones, however, are present in a stoichiometric 1:1:1:1 ratio of H2A:H2B:H3:H4 in the nucleosome. Due to their equal abundance, using a peptide from a single histone for normalization of all measured histone PTMs should generate similar quantification results and relative standard deviation (RSD) without a bias for using the respective histone that contains the PTM for normalization. We performed a series of normalizations, normalizing all measured PTMs to a single histone to evaluate the effect of normalization (Fig. 2a). We were able to show that there is a close consensus in RSD between H2A, H2B, and H3 normalization. H4 normalization varied in RSD with H4 RSD matching that of the other histones or being more or less than that of the other histones used for normalization. While H4 was often different than the consensus RSD for other histones, it did not show bias for H4 PTMs or PTMs of other histones. While the histone used for normalization should not have an effect, we would expect any effect on normalization to have a bias for normalizing PTMs to their respective histone, resulting in lower RSD for H4 PTMs to total H4 pairs. Additionally, no single histone showed a significantly different RSD when used for normalization. The average RSD when PTMs were normalized to their respective histones was 19 %, while normalizing all PTMs to one histone yielded an average RSD of 19 % for H2A, 20 % for H2B, 19 % for H3, and 22 % for H4. Overall, we were not able to observe any bias in histone normalization, which supports the stoichiometric relationship of histones in the nucleosome and the quality of our normalized measurements.Fig. 2


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

Anderson KW, Turko IV - Clin Proteomics (2015)

Histone PTMs in frontal cortex. a Measured peak areas of each quantified peptide were normalized to the peak area of different histone subunits. The relative standard deviation (n = 12) for each quantified peptide in normal and AD frontal cortex is shown grouped by the histone in which the peptides are located. The identity of the PTM measurements is presented in the graph in b, which appears in the same order. b Changes in histone PTMs in AD-affected human frontal cortex. Measurements were performed using human frontal cortex from normal (n = 6) and AD-affected (n = 6) donors (Additional file 1: Table S1) and two experimental replicates per donor (n = 12 total measurements per condition). Q-peptide transitions are summarized in Table 1. Data presented as mean ± SD. PTMs are (ac) acetylation, (me) methylation, and (ub) ubquitination. *p < 0.05; **p < 0.01; ***p < 0.001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Histone PTMs in frontal cortex. a Measured peak areas of each quantified peptide were normalized to the peak area of different histone subunits. The relative standard deviation (n = 12) for each quantified peptide in normal and AD frontal cortex is shown grouped by the histone in which the peptides are located. The identity of the PTM measurements is presented in the graph in b, which appears in the same order. b Changes in histone PTMs in AD-affected human frontal cortex. Measurements were performed using human frontal cortex from normal (n = 6) and AD-affected (n = 6) donors (Additional file 1: Table S1) and two experimental replicates per donor (n = 12 total measurements per condition). Q-peptide transitions are summarized in Table 1. Data presented as mean ± SD. PTMs are (ac) acetylation, (me) methylation, and (ub) ubquitination. *p < 0.05; **p < 0.01; ***p < 0.001
Mentions: Changes in quantified histone PTMs between AD and normal frontal cortex from human donors are presented in Fig. 2b. For quantification of histone PTMs, the peak areas of modified peptides were normalized to the peak areas of non-modified peptides from the respective histone. For example, ubiquitinated H2B peptide AVTKubYTSSK was normalized to H2B peptide EIQTAVR while acetylated H4 peptide GLGKacGGAKacR was normalized to H4 peptide VFLENVIR. While normalization of PTMs within a protein to the total amount of the protein is an accepted practice for quantification [20], it is typically not advisable to normalize PTMs within one protein to the total abundance of a different protein. Histones, however, are present in a stoichiometric 1:1:1:1 ratio of H2A:H2B:H3:H4 in the nucleosome. Due to their equal abundance, using a peptide from a single histone for normalization of all measured histone PTMs should generate similar quantification results and relative standard deviation (RSD) without a bias for using the respective histone that contains the PTM for normalization. We performed a series of normalizations, normalizing all measured PTMs to a single histone to evaluate the effect of normalization (Fig. 2a). We were able to show that there is a close consensus in RSD between H2A, H2B, and H3 normalization. H4 normalization varied in RSD with H4 RSD matching that of the other histones or being more or less than that of the other histones used for normalization. While H4 was often different than the consensus RSD for other histones, it did not show bias for H4 PTMs or PTMs of other histones. While the histone used for normalization should not have an effect, we would expect any effect on normalization to have a bias for normalizing PTMs to their respective histone, resulting in lower RSD for H4 PTMs to total H4 pairs. Additionally, no single histone showed a significantly different RSD when used for normalization. The average RSD when PTMs were normalized to their respective histones was 19 %, while normalizing all PTMs to one histone yielded an average RSD of 19 % for H2A, 20 % for H2B, 19 % for H3, and 22 % for H4. Overall, we were not able to observe any bias in histone normalization, which supports the stoichiometric relationship of histones in the nucleosome and the quality of our normalized measurements.Fig. 2

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