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Quantitative proteomic analysis identifies targets and pathways of a 2-aminobenzamide HDAC inhibitor in Friedreich's ataxia patient iPSC-derived neural stem cells.

Shan B, Xu C, Zhang Y, Xu T, Gottesfeld JM, Yates JR - J. Proteome Res. (2014)

Bottom Line: After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine.The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis.Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, ‡Department of Cell and Molecular biology, The Scripps Research Institute , La Jolla, California 92037, United States.

ABSTRACT
Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

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Related in: MedlinePlus

Photoaffinity labeling of proteins in a nuclearextract from FRDA-iPSC derived neural stem cells with 106 probe followedby addition of a biotin-azide by “click” chemistry,streptavidin capture, and Western blotting with antibody to the indicatedHDACs. Lane 1, nuclear extract input (2% of total, relative to lanes2–3). For HDACs 1 and 3, lane 2, 106-bound proteins; lane 3,control (Ctrl) probe-bound proteins. For HDAC2 western, lane 2, controlprobe-bound proteins; lane 3, 106 probe bound proteins. See ref (7) for detailed methods.
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fig4: Photoaffinity labeling of proteins in a nuclearextract from FRDA-iPSC derived neural stem cells with 106 probe followedby addition of a biotin-azide by “click” chemistry,streptavidin capture, and Western blotting with antibody to the indicatedHDACs. Lane 1, nuclear extract input (2% of total, relative to lanes2–3). For HDACs 1 and 3, lane 2, 106-bound proteins; lane 3,control (Ctrl) probe-bound proteins. For HDAC2 western, lane 2, controlprobe-bound proteins; lane 3, 106 probe bound proteins. See ref (7) for detailed methods.

Mentions: A total of four replicates were performed; 3003proteins were quantified in at least two of the replicates, and thisset was used for further analysis. One thousand two hundred and thirty-oneproteins have an average ratio (ABPP 106 versus control probe) greaterthan 1.5 with a p-value <0.05, and among thoseproteins 883 had an average ratio greater than 2 (Figure 3). HDAC1 and 2 were identified as 106-probe specificbinders and were verified by Western blot analysis (Figure 4). HDAC1 and 2 were found to be significantly enrichedin the ABPP 106 incubated samples.


Quantitative proteomic analysis identifies targets and pathways of a 2-aminobenzamide HDAC inhibitor in Friedreich's ataxia patient iPSC-derived neural stem cells.

Shan B, Xu C, Zhang Y, Xu T, Gottesfeld JM, Yates JR - J. Proteome Res. (2014)

Photoaffinity labeling of proteins in a nuclearextract from FRDA-iPSC derived neural stem cells with 106 probe followedby addition of a biotin-azide by “click” chemistry,streptavidin capture, and Western blotting with antibody to the indicatedHDACs. Lane 1, nuclear extract input (2% of total, relative to lanes2–3). For HDACs 1 and 3, lane 2, 106-bound proteins; lane 3,control (Ctrl) probe-bound proteins. For HDAC2 western, lane 2, controlprobe-bound proteins; lane 3, 106 probe bound proteins. See ref (7) for detailed methods.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227551&req=5

fig4: Photoaffinity labeling of proteins in a nuclearextract from FRDA-iPSC derived neural stem cells with 106 probe followedby addition of a biotin-azide by “click” chemistry,streptavidin capture, and Western blotting with antibody to the indicatedHDACs. Lane 1, nuclear extract input (2% of total, relative to lanes2–3). For HDACs 1 and 3, lane 2, 106-bound proteins; lane 3,control (Ctrl) probe-bound proteins. For HDAC2 western, lane 2, controlprobe-bound proteins; lane 3, 106 probe bound proteins. See ref (7) for detailed methods.
Mentions: A total of four replicates were performed; 3003proteins were quantified in at least two of the replicates, and thisset was used for further analysis. One thousand two hundred and thirty-oneproteins have an average ratio (ABPP 106 versus control probe) greaterthan 1.5 with a p-value <0.05, and among thoseproteins 883 had an average ratio greater than 2 (Figure 3). HDAC1 and 2 were identified as 106-probe specificbinders and were verified by Western blot analysis (Figure 4). HDAC1 and 2 were found to be significantly enrichedin the ABPP 106 incubated samples.

Bottom Line: After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine.The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis.Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology, ‡Department of Cell and Molecular biology, The Scripps Research Institute , La Jolla, California 92037, United States.

ABSTRACT
Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

Show MeSH
Related in: MedlinePlus