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Figure 5. p300/CBP functions as a scaffold, bridge, and acetyltransferase. The acetyltransferase reactions are illustrated by turquoise arrows indicating acetylation of histone and nonhistone substrates (in yellow), as well as autoacetylation of the p300/CBP acetyltransferase domain. The bridge function is illustrated by turquoise squares, representing DNA-binding proteins that bring DNA elements into proximity with p300/CBE through their interactions. The scaffold function is illustrated by orange squares, representing a protein complex being recruited by p300/CBP These functions together allow for gene expression.

Figure 5 p300/CBP functions as a scaffold, bridge, and acetyltransferase. The acetyltransferase reactions are illustrated by turquoise arrows indicating acetylation of histone and nonhistone substrates (in yellow), as well as autoacetylation of the p300/CBP acetyltransferase domain. The bridge function is illustrated by turquoise squares, representing DNA-binding proteins that bring DNA elements into proximity with p300/CBE through their interactions. The scaffold function is illustrated by orange squares, representing a protein complex being recruited by p300/CBP These functions together allow for gene expression.

Related Figures (21)

Figure 1. Reversible acetylation of the epsilon amine group of lysine side chains. This Review is focused on lysine acetylation. The acetyl group in question is donated by the cofactor, acetyl-coenzyme A. Coenzyme A, which is found in all characterized living organisms on earth,” is synthesized by cells from pantothenic acid (vitamin BS) and cysteine,” and its thiol moiety can react with the carboxylic acids of acyl groups to activate those groups with an energy-rich thioester bond. Acetyltransferase enzymes catalyze the covalent transfer of the acetyl group from acetyl- CoA to the epsilon amine of the lysine side chain, with the release of the free thiol, CoASH (see Figure 1). The resultant
Figure 1. Reversible acetylation of the epsilon amine group of lysine side chains. This Review is focused on lysine acetylation. The acetyl group in question is donated by the cofactor, acetyl-coenzyme A. Coenzyme A, which is found in all characterized living organisms on earth,” is synthesized by cells from pantothenic acid (vitamin BS) and cysteine,” and its thiol moiety can react with the carboxylic acids of acyl groups to activate those groups with an energy-rich thioester bond. Acetyltransferase enzymes catalyze the covalent transfer of the acetyl group from acetyl- CoA to the epsilon amine of the lysine side chain, with the release of the free thiol, CoASH (see Figure 1). The resultant
Figure 2. Major metabolic processes that produce or consume acetyl- CoA. Processes occurring in the cytoplasm are indicated using purple font, and processes occurring in the mitochondrion are indicated using orange font. Note that PDH can also be nuclear. This figure was adapted in part from Albaugh et al.°°
Figure 2. Major metabolic processes that produce or consume acetyl- CoA. Processes occurring in the cytoplasm are indicated using purple font, and processes occurring in the mitochondrion are indicated using orange font. Note that PDH can also be nuclear. This figure was adapted in part from Albaugh et al.°°
Figure 3. Domain structure of p300/CBP. Exon—intron gene diagrams are shown for p300 and CBP (top). Below are example protein structures for the bromodomain (PDB 313J, 2.33 A), catalytic HAT domain (PDB 3BIY, 1.7 A), ZZ zinc finger (PDB 1TOT), and TAZ2 domain (PDB 3102, 2.5 A). All structures were produced using purified p300, except the ZZ zinc finger, which used purified CBP. p300/CBP proteins are colored with a rainbow, with blue at the N-terminus and red at the C-terminus, and residues included in the structure are listed below each. Zinc ions are black spheres. All structures are based on X-ray crystallography, except the ZZ zinc finger structures from solution NMR. The p300 bromodomain structure shown here is remarkably similar to an independently generated CBP bromodomain structure (not shown, PDB 3DWY, 1.98 A). Below is a model for full-length p300/CBP with all domains shown, and is a compilation based on several recent analyses.:”*”° three cysteine/histidine-rich (C/ H) domains are shown in turquoise, three zinc fingers are shown in yellow, and the catalytic acetyltransferase domain is shown in orange, with its autoacetylated regulatory loop drawn above, which corresponds to residues 1523—1554. A few examples of proteins that bind p300/CBP are listed below the protein model, with the particular domain involved in binding indicated with a black line. Below that, amino acid similarity is indicated, for comparing p300 and CBP sequences within either the catalytic BHC region (from the bromodomain to the C/H3 domain) or the entire protein. At the bottom, commonly purified active p300 variants are indicated, including p300 acetyltransferase/HAT domain, BHC enzyme (bromodomain- HAT-C/H3), and full-length protein. It should be noted that p300 HAT has a deletion in residues 1529-1560.
Figure 3. Domain structure of p300/CBP. Exon—intron gene diagrams are shown for p300 and CBP (top). Below are example protein structures for the bromodomain (PDB 313J, 2.33 A), catalytic HAT domain (PDB 3BIY, 1.7 A), ZZ zinc finger (PDB 1TOT), and TAZ2 domain (PDB 3102, 2.5 A). All structures were produced using purified p300, except the ZZ zinc finger, which used purified CBP. p300/CBP proteins are colored with a rainbow, with blue at the N-terminus and red at the C-terminus, and residues included in the structure are listed below each. Zinc ions are black spheres. All structures are based on X-ray crystallography, except the ZZ zinc finger structures from solution NMR. The p300 bromodomain structure shown here is remarkably similar to an independently generated CBP bromodomain structure (not shown, PDB 3DWY, 1.98 A). Below is a model for full-length p300/CBP with all domains shown, and is a compilation based on several recent analyses.:”*”° three cysteine/histidine-rich (C/ H) domains are shown in turquoise, three zinc fingers are shown in yellow, and the catalytic acetyltransferase domain is shown in orange, with its autoacetylated regulatory loop drawn above, which corresponds to residues 1523—1554. A few examples of proteins that bind p300/CBP are listed below the protein model, with the particular domain involved in binding indicated with a black line. Below that, amino acid similarity is indicated, for comparing p300 and CBP sequences within either the catalytic BHC region (from the bromodomain to the C/H3 domain) or the entire protein. At the bottom, commonly purified active p300 variants are indicated, including p300 acetyltransferase/HAT domain, BHC enzyme (bromodomain- HAT-C/H3), and full-length protein. It should be noted that p300 HAT has a deletion in residues 1529-1560.
Figure 4. p300/CBP is central to many important signaling pathways. These include pathways that respond to intracellular signals (turquoise), extracellular signals (purple), and intercellular signals (blue). These pathways control the key cellular functions via altering expression of target genes, through the action of p300/CBP in the nucleus.
Figure 4. p300/CBP is central to many important signaling pathways. These include pathways that respond to intracellular signals (turquoise), extracellular signals (purple), and intercellular signals (blue). These pathways control the key cellular functions via altering expression of target genes, through the action of p300/CBP in the nucleus.
Figure 7. Acetyl transfer catalysis by p300. (A) The p300 active site is drawn in green, and histone H4 substrate in blue, with important residues indicated. CoA is drawn in black, and binds in a specific tunnel. (B) Four steps in a proposed p300 mechanism. acetyl-CoA binds, then peptidyl- lysine binds. The hydrophobic indole of W1436 promotes an uncharged lysine and positions it for attack. The lysine attacks the carbonyl of acetyl- CoA, while Y1467 acts as a general acid to protonate the leaving group. Acetyl-lysine-containing product leaves quickly, then CoASH departs slowly.
Figure 7. Acetyl transfer catalysis by p300. (A) The p300 active site is drawn in green, and histone H4 substrate in blue, with important residues indicated. CoA is drawn in black, and binds in a specific tunnel. (B) Four steps in a proposed p300 mechanism. acetyl-CoA binds, then peptidyl- lysine binds. The hydrophobic indole of W1436 promotes an uncharged lysine and positions it for attack. The lysine attacks the carbonyl of acetyl- CoA, while Y1467 acts as a general acid to protonate the leaving group. Acetyl-lysine-containing product leaves quickly, then CoASH departs slowly.
The first potent acetyltransferase inhibitor identified is Lys- CoA,'** which is a covalent fusion between an acetyl-lysine and coenzyme A, with an extra methylene in between (see Figure 8). Structure—activity relationship studies have subsequently Figure 8. Bisubstrate inhibitors of acetyltransferases.
The first potent acetyltransferase inhibitor identified is Lys- CoA,'** which is a covalent fusion between an acetyl-lysine and coenzyme A, with an extra methylene in between (see Figure 8). Structure—activity relationship studies have subsequently Figure 8. Bisubstrate inhibitors of acetyltransferases.
Figure 9. Natural products implicated as modulators of acetyltrans- ferases.
Figure 9. Natural products implicated as modulators of acetyltrans- ferases.
Figure 10. Synthetic inhibitors of acetyltransferases.
Figure 10. Synthetic inhibitors of acetyltransferases.
Figure 11. C646 modeled in the acetyltransferase active site. (A) C646 is shown in magenta, computationally docked in the crystal structure of th acetyltransferase active site, which was generated as a cocrystal with Lys-CoA. Several residues that coordinate CoA binding are predicted to similarh coordinate C646 binding, as shown in aqua stick representations of the side chains. (B) The structure of C646, shown in an orientation similar t« that in the docked model above.
Figure 11. C646 modeled in the acetyltransferase active site. (A) C646 is shown in magenta, computationally docked in the crystal structure of th acetyltransferase active site, which was generated as a cocrystal with Lys-CoA. Several residues that coordinate CoA binding are predicted to similarh coordinate C646 binding, as shown in aqua stick representations of the side chains. (B) The structure of C646, shown in an orientation similar t« that in the docked model above.
Figure 13. Structures of p300/CBP TAZ2 domain bound to ligands. The TAZ2 domain is shown colored in a rainbow (blue to red, residues included listed below each) bound to various ligands: STAT 1 (A, PDB 2KA6); E1A (B, PDB 2KJE); p53 (C, PDB 2K8F); MEF2- DNA complex (D, PDB 3P57), and C/EBPe (PDB 3T92). All structures are based on solution NMR except for two from X-ray crystallography: that in (D) (2.192 A) and that in (E) (1.5 A). All structures were produced using purified p300, except the (A) and (B), which used purified CBP. Zinc ions are black spheres, protein ligands are gray, and DNA is yellow. The crystal structure with MEF2 revealed binding in three possible conformations with TAZ2, and one example is shown here.
Figure 13. Structures of p300/CBP TAZ2 domain bound to ligands. The TAZ2 domain is shown colored in a rainbow (blue to red, residues included listed below each) bound to various ligands: STAT 1 (A, PDB 2KA6); E1A (B, PDB 2KJE); p53 (C, PDB 2K8F); MEF2- DNA complex (D, PDB 3P57), and C/EBPe (PDB 3T92). All structures are based on solution NMR except for two from X-ray crystallography: that in (D) (2.192 A) and that in (E) (1.5 A). All structures were produced using purified p300, except the (A) and (B), which used purified CBP. Zinc ions are black spheres, protein ligands are gray, and DNA is yellow. The crystal structure with MEF2 revealed binding in three possible conformations with TAZ2, and one example is shown here.
Figure 14. Models for targeting influenced by p300-ligand binding. In these models, p300 is shown in green, the histone octamer is shown in yellow, DNA is shown with a red strand, and p300 ligands are indicated with an “L”. In (A), a ligand targets p300 to a gene or other DNA element due to the DNA binding affinity of the ligand. In (B), a ligand targets p300 to a protein complex due to the protein binding affinity of the ligand. In (C), two ligands bridged by p300 allow for chromatin (bound by the purple ligand) to come into proximity with a chromatin-modifying enzyme (the orange ligand). In (D), two ligands compete for the same site within p300, and the one ligand could be seen as a competitive inhibitor for the p300 association with the other.
Figure 14. Models for targeting influenced by p300-ligand binding. In these models, p300 is shown in green, the histone octamer is shown in yellow, DNA is shown with a red strand, and p300 ligands are indicated with an “L”. In (A), a ligand targets p300 to a gene or other DNA element due to the DNA binding affinity of the ligand. In (B), a ligand targets p300 to a protein complex due to the protein binding affinity of the ligand. In (C), two ligands bridged by p300 allow for chromatin (bound by the purple ligand) to come into proximity with a chromatin-modifying enzyme (the orange ligand). In (D), two ligands compete for the same site within p300, and the one ligand could be seen as a competitive inhibitor for the p300 association with the other.
Figure 15. Mastermind-Notch-CSL-DNA core complex. The complex formed by DNA, CSL, Notch (ANK repeats and RAM region purified separately), and Mastermind N-terminal helix is shown with two different view angles. The X-ray crystal structure was generated at 3.85 A, and this figure was produced in PyMol using Protein Databank entry 3V79. Proteins are shown as ribbons, with the surfaces at 70% transparency. DNA is shown as stick models colored by atom.
Figure 15. Mastermind-Notch-CSL-DNA core complex. The complex formed by DNA, CSL, Notch (ANK repeats and RAM region purified separately), and Mastermind N-terminal helix is shown with two different view angles. The X-ray crystal structure was generated at 3.85 A, and this figure was produced in PyMol using Protein Databank entry 3V79. Proteins are shown as ribbons, with the surfaces at 70% transparency. DNA is shown as stick models colored by atom.
Figure 16. Model for Mastermind activation of p300. In this model, p300 (green) initially has inhibited acetyltransferase activity due to an autoinhibitory loop (orange, left). This is relieved upon recruitment by the Notch-Mastermind-CSL complex. Mastermind (purple) binds to the p300 C/H3 domain, and also to Notch intracellular domain (magenta) and CSL (red). p300 autoacetylation, Mastermind acetylation, and histone acetylation are then catalyzed by p300 (turquoise A).
Figure 16. Model for Mastermind activation of p300. In this model, p300 (green) initially has inhibited acetyltransferase activity due to an autoinhibitory loop (orange, left). This is relieved upon recruitment by the Notch-Mastermind-CSL complex. Mastermind (purple) binds to the p300 C/H3 domain, and also to Notch intracellular domain (magenta) and CSL (red). p300 autoacetylation, Mastermind acetylation, and histone acetylation are then catalyzed by p300 (turquoise A).
Figure 17. Inhibitors of the p300/CBP bromodomain.
Figure 17. Inhibitors of the p300/CBP bromodomain.
proteins known to also interact with transactivation domains, such as TRRAP and Med23. Binding of iTAD to KIX competitively inhibits endogenous transactivation domain- containing ligands from binding the same site, while activating CBP activities that are normally induced by protein ligand binding.**? The addition of a DNA-binding moiety to iTAD can allow activation of the associated gene by recruiting the active CBP.*° The CBP KIX domain may also be the target of the compound ICG-001 (see Figure 18), which appears to block the association of CBP with /-catenin, and decrease expression of genes usually induced in the Wnt//-catenin/CBP pathway. Although the specificity of this compound is not known, nor the exact binding mechanism, it does show promising effects in animal models of colon cancer, myocardial infarction, pulmonary fibrosis, and fibrotic kidney dis- 361-364 ease.
proteins known to also interact with transactivation domains, such as TRRAP and Med23. Binding of iTAD to KIX competitively inhibits endogenous transactivation domain- containing ligands from binding the same site, while activating CBP activities that are normally induced by protein ligand binding.**? The addition of a DNA-binding moiety to iTAD can allow activation of the associated gene by recruiting the active CBP.*° The CBP KIX domain may also be the target of the compound ICG-001 (see Figure 18), which appears to block the association of CBP with /-catenin, and decrease expression of genes usually induced in the Wnt//-catenin/CBP pathway. Although the specificity of this compound is not known, nor the exact binding mechanism, it does show promising effects in animal models of colon cancer, myocardial infarction, pulmonary fibrosis, and fibrotic kidney dis- 361-364 ease.
While loss-of-function in p300/CBP is deleterious, gain-of- function in p300/CBP also contributes to human disease, because the correct balance of acetylation and deacetylation, in the right time and place, is required for normal cell functioning. This section explores cases of gain-of-function in p300/CBP, either by direct mutation or overexpression of p300/CBP or by the involvement of p300/CBP in the disease etiology, as well as additional instances where disruption of p300/CBP activity would be therapeutically advantageous (see Figure 19). Figure 19. Diseases of potential therapeutic application for a p300/ CBP inhibitor. No p300/CBP inhibitor has yet made it into clinical trials, but the biology of p300/CBP action and documented effects of p300/CBP disruption lead us to hypothesize a beneficial therapeutic potential for a p300/CBP inhibitor in many diseases.
While loss-of-function in p300/CBP is deleterious, gain-of- function in p300/CBP also contributes to human disease, because the correct balance of acetylation and deacetylation, in the right time and place, is required for normal cell functioning. This section explores cases of gain-of-function in p300/CBP, either by direct mutation or overexpression of p300/CBP or by the involvement of p300/CBP in the disease etiology, as well as additional instances where disruption of p300/CBP activity would be therapeutically advantageous (see Figure 19). Figure 19. Diseases of potential therapeutic application for a p300/ CBP inhibitor. No p300/CBP inhibitor has yet made it into clinical trials, but the biology of p300/CBP action and documented effects of p300/CBP disruption lead us to hypothesize a beneficial therapeutic potential for a p300/CBP inhibitor in many diseases.
Biographies Beverley M. Dancy, originally from Cape Town, was introduced to research in the laboratory of Greg J. Hermann at Lewis & Clark College. Her graduate studies with Philip A. Cole, centered on p300 acetyltransferase inhibition, earned her a Ph.D. in Pharmacology and Molecular Sciences from the Johns Hopkins University School of Medicine in 2013. As a postdoctoral fellow in the Morgan-Sedensky laboratory, she investigated enzymes and pathways involved in mitochondrial disease, and in 2014 she will begin a fellowship at the National Institutes of Health Laboratory of Cardiac Energetics, with Robert S. Balaban. Dr. Dancy is interested in developing molecular The authors declare the following competing financial interest(s): P.A.C. is a cofounder, advisor, and equity owner in the company Acylin Therapeutics that is developing HAT inhibitors. B.M.D. declares no competing financial interest.
Biographies Beverley M. Dancy, originally from Cape Town, was introduced to research in the laboratory of Greg J. Hermann at Lewis & Clark College. Her graduate studies with Philip A. Cole, centered on p300 acetyltransferase inhibition, earned her a Ph.D. in Pharmacology and Molecular Sciences from the Johns Hopkins University School of Medicine in 2013. As a postdoctoral fellow in the Morgan-Sedensky laboratory, she investigated enzymes and pathways involved in mitochondrial disease, and in 2014 she will begin a fellowship at the National Institutes of Health Laboratory of Cardiac Energetics, with Robert S. Balaban. Dr. Dancy is interested in developing molecular The authors declare the following competing financial interest(s): P.A.C. is a cofounder, advisor, and equity owner in the company Acylin Therapeutics that is developing HAT inhibitors. B.M.D. declares no competing financial interest.
tools and imaging technologies for understanding the consequences of disease at the molecular, cellular, and organismal levels. In addition to research, she is passionate about bridging the gap between science research and a broader audience through communication and engagement. Philip A. Cole was born in Paterson, NJ and graduated from Yale University with a B.S. in Chemistry in 1984 and then spent a year as a Churchill Scholar at the University of Cambridge, England. Cole went on to obtain M.D. and Ph.D. degrees from Johns Hopkins University where he pursued research in bioorganic chemistry in 1991. Cole then entered postdoctoral fellowship training at Harvard Medical School prior to joining the Rockefeller University in 1996 as a junior lab head. In 1999, Cole moved back to Johns Hopkins as the Marshall-Maren professor and director of pharmacology. His research interests are in the area of protein post-translational modifications and chemical biology.
tools and imaging technologies for understanding the consequences of disease at the molecular, cellular, and organismal levels. In addition to research, she is passionate about bridging the gap between science research and a broader audience through communication and engagement. Philip A. Cole was born in Paterson, NJ and graduated from Yale University with a B.S. in Chemistry in 1984 and then spent a year as a Churchill Scholar at the University of Cambridge, England. Cole went on to obtain M.D. and Ph.D. degrees from Johns Hopkins University where he pursued research in bioorganic chemistry in 1991. Cole then entered postdoctoral fellowship training at Harvard Medical School prior to joining the Rockefeller University in 1996 as a junior lab head. In 1999, Cole moved back to Johns Hopkins as the Marshall-Maren professor and director of pharmacology. His research interests are in the area of protein post-translational modifications and chemical biology.
Figure 17. Inhibitors of the p300/CBP bromodomain. Correction to Protein Lysine Acetylation by p300/CBP
Figure 17. Inhibitors of the p300/CBP bromodomain. Correction to Protein Lysine Acetylation by p300/CBP
Related topics:
Chromatin Remodeling and EpigeneticsHistone ModificationsFRET microscopyGene expression and regulation
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