news & views
H 2N HN
NH 2
Arginine
PAD1, 2, 3, 4, 6 Ca2+
H2O
NH3
H 2N
Upregulation of pluripotency markers O
Decondensed chromatin
HN
Klf2
Tcfap2c
Tcl1
Kit
Nanog
H3Cit
Citrulline
PAD4
H1Cit
Upregulation
RNA POL II
PAD4 Inhibition (Cl-amidine, TDFA)
npg
© 2014 Nature America, Inc. All rights reserved.
DNA
Core histones
Linker histone H1
Compact chromatin
Prickle1
Epha1
Wnt8a
Upregulation of differentiation markers
Figure 1 | PAD4 is a regulator of pluripotency gene expression through the conversion of arginine to citrulline in histones. PAD4 citrullinates core (H3 and H4) and linker (H1) histones, leading to chromatin decondensation and the expression of pluripotency markers in embryonic stem cells, which can be reversed upon inhibition of PAD activity.
The expression of pluripotency genes was also found to be dependent on PAD4 enzymatic activity, as inhibition with the pan-PAD inhibitor Cl-amidine9 and the PAD4-selective inhibitor TDFA10 reduced citrullinated H3 (H3Cit), which in turn reduced the expression of the pluripotency genes Nanog, Tcl1 and Klf5. Inhibition of PAD4 activity also led to increased expression of differentiation genes, including Prickle1, Epha1 and Wnt8a, and stem cells treated with TDFA reduced the number of pluripotent cells in early embryogenesis. These results were validated by RNAi knockdown of PAD4. To further investigate the role of PAD4 in pluripotency, Christophorou et al.8 identified several citrullinated proteins, including AtrX, Dnmt3b, Trim28 and histone H1, all of which help to control the pluripotent state. Importantly, histones H1.2, H1.3, H1.4 and H1.5
were citrullinated in the central winged helix DNA-binding domain at Arg54 (H1R54Cit), and mutation of this residue (R54A) resulted in the release of H1 from chromatin. Inhibition of PAD4 expression or activity also decreased histone citrullination and favored a compacted chromatin state, which correlated with the downregulation of pluripotency genes and the upregulation of differentiation genes. Interestingly, there are parallels to neutrophil extracellular trap formation where chromatin decondensation is driven by the PAD4-mediated citrullination of both histone H1 and H3, and the site of H1 citrullination is the same as that observed in pluripotent stem cells7. In summary, this work (Fig. 1) adds to our growing understanding of PAD biology in a variety of physiological and pathophysiological processes and furthermore reiterates the value in
developing selective and potent chemical probes to pharmacologically modulate enzymes, such as the PADs, in cellular systems. These studies also raise a number of important issues that will undoubtedly fuel future studies. First, although the authors focused on PAD4, they also observed increased expression of PADs 1, 2 and 3 in ES and/or iPS cells, which suggest that multiple PADs could have a role in pluripotency or function at different stages during cellular differentiation. Second, PADs 1–4 are overexpressed in multiple cancers, and it seems likely that their activity may ‘reprogram’ cancer cells into a more stem cell–like state and thereby promote their unrestrained growth. Third, as interest in the PADs as therapeutic targets for cancer and inflammatory diseases continues to increase, some caution is warranted as drugs targeting these enzymes may affect stem cell fate and embryogenesis. Nevertheless, the future of PAD biology is bright and represents a promising area of growth in our understanding of the delicate balance between health and disease. ■ Daniel J. Slade, Venkataraman Subramanian and Paul R. Thompson are at the Scripps Research Institute, Department of Chemistry, Jupiter, Florida, USA. e-mail: [email protected] References
1. Bicker, K.L. & Thompson, P.R. Biopolymers 99, 155–163 (2013). 2. Kaplan, M.J. & Radic, M. J. Immunol. 189, 2689–2695 (2012). 3. Cherrington, B.D. et al. PLoS ONE 7, e41242 (2012). 4. Cuthbert, G.L. et al. Cell 118, 545–553 (2004). 5. Yao, H. et al. J. Biol. Chem. 283, 20060–20068 (2008). 6. Sharma, P. et al. PLoS Genet. 8, e1002934 (2012). 7. Dwivedi, N. et al. FASEB J. doi:10.1096/fj.13-247254 (2014). 8. Christophorou, M.A. et al. Nature 507, 104–108 (2014). 9. Luo, Y. et al. Biochemistry 45, 11727–11736 (2006). 10. Jones, J.E. et al. ACS Chem. Biol. 7, 160–165 (2012).
Competing financial interests
The authors declare competing financial interests: details accompany the online version of the paper.
LABELING
Palladium brings proteins to life
A bioorthogonal decaging strategy, mediated by small-molecule palladium compounds, can recover the lysinedependent activity of cellular proteins. This activation technique could be generally applicable for controlling and probing function of a protein target in living cells.
Kun Qian & Yujun George Zheng
T
he creation of small-molecule effectors and other biologically applicable tools to specifically control (either inhibit or activate) a protein of interest (POI) with 328
spatial and temporal resolution, especially in living cells, continues to be a challenging theme in chemical biology and drug discovery 1,2. Chemical modulation
complements classical genetic methods to interrogate biology by providing a powerful means to decipher the function of a specific gene or gene product with greater dynamic
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
news & views
OspF secretion Nucleus
OspF (Off)
O
[Pd]
HN
OspF (On)
O
K134 N
H
O
H
–
OPO3
H
O N H
npg
© 2014 Nature America, Inc. All rights reserved.
–
Erk
H
H2N+
H
O N H
O3PO
Erk
Dephosphorylation (elimination)
Import/export (reversible)
Export only (irreversible)
OH
Erk
Erk
(Cytosolic accumulation) Figure 1 | Palladium-triggered chemical rescue for protein activation in living cells. The inactive form of bacterial secretion effector OspF (ProcLys134) is activated by a bioorthogonal palladium-mediated deprotection reaction. The activated OspF catalyzes the irreversible dephosphorylation of phosphorylated Erk, leading to accumulation of the damaged Erk substrate in the cytosol of the host cell and causing an imbalance in Erk’s nucleus-cytoplasm equilibrium. The propargyloxycarbonyl-protected lysine is highlighted in red.
control (by varying dose, timing and location). Although the inhibitor-based, loss-of-function approach has been more widely adopted, small-molecule activators that promote or turn on protein activity in a gain-of-function fashion have unique advantages. This is especially true for those protein targets that can produce amplifiable biological readouts such as enzymes and transcription factors. Because of the dazzling diversity of protein structures, there exists no single universal strategy for activating POIs with small molecules, and most available probes were identified by unbiased library screening or serendipitous finding3,4. One recognized strategy is chemical rescue, in which a mutation is introduced in the POI to silence its nascent activity (i.e., creating a dormant state), and then an exogenous ligand is applied to complement the defect caused by the mutation, thus (at least partially) rescuing the wild-type activity of the protein target. Though this method proved to be effective in several case studies5, it is difficult to generalize because of structural variation across proteins.
Li et al.6 developed a different approach to the idea of ‘chemical rescue’ in their report of a chemical decaging strategy that can be used to specifically activate a POI (Fig. 1). The method relies on the use of a small-molecule palladium catalyst to remove an unsaturated protecting group (allyl or propargyl) from lysine. In recent years, application of transition metal-based catalysts in living biological systems has drawn great attention. Allyl and propargyl groups have superior bioorthogonality and unique reactivity to undergo tailored chemical transformations (for example, cleavage and cross-coupling) and so are ideal targets for selective catalysis by organometallic complexes7,8. In their report, Li et al.6 first screened a focused library of possible palladium catalysts, finding that propargyl carbamate (Proc) and, to a lesser extent, allyl carbamate could be effectively removed from lysine using biocompatible catalysts such as Pd(dba)2 and Allyl2Pd2Cl2. The authors validated the reaction on model compounds and purified proteins before turning to more
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
complex environments, in which Proc-lysine was site-specifically incorporated into a protein target using the genetically encoded unnatural amino acid insertion method9. To make optimal use of the caged lysine residue, Li et al.6 focused on proteins in which lysine residues are critical for activity, for example, nearby or at the active site of an enzyme. In this way, the wild-type activity of the caged protein would remain dormant until the palladium reagent–mediated Proc cleavage unleashed the free ε-amine of the lysine. As a case study, the authors expressed OspF, a phosphothreonine lyase, with Proclysine incorporated at a critical position (K134) such that its enzymatic activity was switched off. The subsequent addition of a membrane-permeable palladium reagent drove elimination of the Proc caging group, restoring OspF activity. With this tool, the authors confirmed that the selected lysine residue was essential for OspF activity and demonstrated that OspF altered the subcellular localization of its substrate, phosphorylated Erk (Fig. 1), explaining how OspF functions to impair cellular signaling pathways as part of bacterial infection. The method developed by Li et al.6 provides a small molecule–mediated chemical biology strategy for activating proteins by regenerating the nascent form of an embedded lysine residue. As the unnatural amino acid coding protocol for lysine replacement by Proc-lysine has been well established9, this new chemical rescue method can, in principle, be applied to any POI that contains a functionally important lysine. Such generality is important as currently there are few other general methods to discover small-molecule protein activators. Given the prevalence of lysine in regulating protein structure, stability, enzyme activity and protein-protein interactions, it is foreseeable that this lysine-dependent activation technique will find great use in elucidating the biological function of a protein target in a specific signaling pathway. In addition, this will be a great tool for studying catalytic roles of lysine residues in enzyme systems. The new strategy may also extend beyond lysines, as propargyl and allyl groups can be readily applied to protect the side chain of any amino acid that contains a nucleophilic atom, including lysine as well as tyrosine, serine, threonine, aspartate, glutamate and so on. However, further research will be needed to demonstrate whether the method can be expanded to these amino acids, as (i) the propargyl or allyl protecting group must be chemically stable within a certain timeframe, (ii) propargyl- or allyl-protected amino acids must be amenable to site-specific 329
news & views incorporation using either the genetic code expansion method or other protein engineering techniques10 and (iii) decaging by a cell-permeable biocompatible organometallic reagent must be rapid and efficient. Future investigations will prove (or disprove) these possibilities. ■
References
1. Schreiber, S.L. Nat. Chem. Biol. 1, 64–66 (2005). 2. Carlson, S.M. & White, F.M. ACS Chem. Biol. 6, 75–85 (2011). 3. Zorn, J.A. & Wells, J.A. Nat. Chem. Biol. 6, 179–188 (2010). 4. Bishop, A.C. & Chen, V.L. J. Chem. Biol. 2, 1–9 (2009). 5. Tarrant, M.K. & Cole, P.A. Annu. Rev. Biochem. 78, 797–825 (2009).
6. Li, J. et al. Nat. Chem. 6, 352–361 (2014). 7. Santra, M., Ko, S.K., Shin, I. & Ahn, K.H. Chem. Commun. (Camb.) 46, 3964–3966 (2010). 8. Garner, A.L., Song, F. & Koide, K. J. Am. Chem. Soc. 131, 5163–5171 (2009). 9. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010). 10. Muralidharan, V. & Muir, T.W. Nat. Methods 3, 429–438 (2006).
Competing financial interests
The authors declare no competing financial interests.
npg
© 2014 Nature America, Inc. All rights reserved.
Kun Qian and Yujun George Zheng are at the Department of Pharmaceutical and Biomedical
Sciences, University of Georgia, Athens, Georgia, USA. e-mail: [email protected]
330
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
H 2N HN
NH 2
Arginine
PAD1, 2, 3, 4, 6 Ca2+
H2O
NH3
H 2N
Upregulation of pluripotency markers O
Decondensed chromatin
HN
Klf2
Tcfap2c
Tcl1
Kit
Nanog
H3Cit
Citrulline
PAD4
H1Cit
Upregulation
RNA POL II
PAD4 Inhibition (Cl-amidine, TDFA)
npg
© 2014 Nature America, Inc. All rights reserved.
DNA
Core histones
Linker histone H1
Compact chromatin
Prickle1
Epha1
Wnt8a
Upregulation of differentiation markers
Figure 1 | PAD4 is a regulator of pluripotency gene expression through the conversion of arginine to citrulline in histones. PAD4 citrullinates core (H3 and H4) and linker (H1) histones, leading to chromatin decondensation and the expression of pluripotency markers in embryonic stem cells, which can be reversed upon inhibition of PAD activity.
The expression of pluripotency genes was also found to be dependent on PAD4 enzymatic activity, as inhibition with the pan-PAD inhibitor Cl-amidine9 and the PAD4-selective inhibitor TDFA10 reduced citrullinated H3 (H3Cit), which in turn reduced the expression of the pluripotency genes Nanog, Tcl1 and Klf5. Inhibition of PAD4 activity also led to increased expression of differentiation genes, including Prickle1, Epha1 and Wnt8a, and stem cells treated with TDFA reduced the number of pluripotent cells in early embryogenesis. These results were validated by RNAi knockdown of PAD4. To further investigate the role of PAD4 in pluripotency, Christophorou et al.8 identified several citrullinated proteins, including AtrX, Dnmt3b, Trim28 and histone H1, all of which help to control the pluripotent state. Importantly, histones H1.2, H1.3, H1.4 and H1.5
were citrullinated in the central winged helix DNA-binding domain at Arg54 (H1R54Cit), and mutation of this residue (R54A) resulted in the release of H1 from chromatin. Inhibition of PAD4 expression or activity also decreased histone citrullination and favored a compacted chromatin state, which correlated with the downregulation of pluripotency genes and the upregulation of differentiation genes. Interestingly, there are parallels to neutrophil extracellular trap formation where chromatin decondensation is driven by the PAD4-mediated citrullination of both histone H1 and H3, and the site of H1 citrullination is the same as that observed in pluripotent stem cells7. In summary, this work (Fig. 1) adds to our growing understanding of PAD biology in a variety of physiological and pathophysiological processes and furthermore reiterates the value in
developing selective and potent chemical probes to pharmacologically modulate enzymes, such as the PADs, in cellular systems. These studies also raise a number of important issues that will undoubtedly fuel future studies. First, although the authors focused on PAD4, they also observed increased expression of PADs 1, 2 and 3 in ES and/or iPS cells, which suggest that multiple PADs could have a role in pluripotency or function at different stages during cellular differentiation. Second, PADs 1–4 are overexpressed in multiple cancers, and it seems likely that their activity may ‘reprogram’ cancer cells into a more stem cell–like state and thereby promote their unrestrained growth. Third, as interest in the PADs as therapeutic targets for cancer and inflammatory diseases continues to increase, some caution is warranted as drugs targeting these enzymes may affect stem cell fate and embryogenesis. Nevertheless, the future of PAD biology is bright and represents a promising area of growth in our understanding of the delicate balance between health and disease. ■ Daniel J. Slade, Venkataraman Subramanian and Paul R. Thompson are at the Scripps Research Institute, Department of Chemistry, Jupiter, Florida, USA. e-mail: [email protected] References
1. Bicker, K.L. & Thompson, P.R. Biopolymers 99, 155–163 (2013). 2. Kaplan, M.J. & Radic, M. J. Immunol. 189, 2689–2695 (2012). 3. Cherrington, B.D. et al. PLoS ONE 7, e41242 (2012). 4. Cuthbert, G.L. et al. Cell 118, 545–553 (2004). 5. Yao, H. et al. J. Biol. Chem. 283, 20060–20068 (2008). 6. Sharma, P. et al. PLoS Genet. 8, e1002934 (2012). 7. Dwivedi, N. et al. FASEB J. doi:10.1096/fj.13-247254 (2014). 8. Christophorou, M.A. et al. Nature 507, 104–108 (2014). 9. Luo, Y. et al. Biochemistry 45, 11727–11736 (2006). 10. Jones, J.E. et al. ACS Chem. Biol. 7, 160–165 (2012).
Competing financial interests
The authors declare competing financial interests: details accompany the online version of the paper.
LABELING
Palladium brings proteins to life
A bioorthogonal decaging strategy, mediated by small-molecule palladium compounds, can recover the lysinedependent activity of cellular proteins. This activation technique could be generally applicable for controlling and probing function of a protein target in living cells.
Kun Qian & Yujun George Zheng
T
he creation of small-molecule effectors and other biologically applicable tools to specifically control (either inhibit or activate) a protein of interest (POI) with 328
spatial and temporal resolution, especially in living cells, continues to be a challenging theme in chemical biology and drug discovery 1,2. Chemical modulation
complements classical genetic methods to interrogate biology by providing a powerful means to decipher the function of a specific gene or gene product with greater dynamic
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
news & views
OspF secretion Nucleus
OspF (Off)
O
[Pd]
HN
OspF (On)
O
K134 N
H
O
H
–
OPO3
H
O N H
npg
© 2014 Nature America, Inc. All rights reserved.
–
Erk
H
H2N+
H
O N H
O3PO
Erk
Dephosphorylation (elimination)
Import/export (reversible)
Export only (irreversible)
OH
Erk
Erk
(Cytosolic accumulation) Figure 1 | Palladium-triggered chemical rescue for protein activation in living cells. The inactive form of bacterial secretion effector OspF (ProcLys134) is activated by a bioorthogonal palladium-mediated deprotection reaction. The activated OspF catalyzes the irreversible dephosphorylation of phosphorylated Erk, leading to accumulation of the damaged Erk substrate in the cytosol of the host cell and causing an imbalance in Erk’s nucleus-cytoplasm equilibrium. The propargyloxycarbonyl-protected lysine is highlighted in red.
control (by varying dose, timing and location). Although the inhibitor-based, loss-of-function approach has been more widely adopted, small-molecule activators that promote or turn on protein activity in a gain-of-function fashion have unique advantages. This is especially true for those protein targets that can produce amplifiable biological readouts such as enzymes and transcription factors. Because of the dazzling diversity of protein structures, there exists no single universal strategy for activating POIs with small molecules, and most available probes were identified by unbiased library screening or serendipitous finding3,4. One recognized strategy is chemical rescue, in which a mutation is introduced in the POI to silence its nascent activity (i.e., creating a dormant state), and then an exogenous ligand is applied to complement the defect caused by the mutation, thus (at least partially) rescuing the wild-type activity of the protein target. Though this method proved to be effective in several case studies5, it is difficult to generalize because of structural variation across proteins.
Li et al.6 developed a different approach to the idea of ‘chemical rescue’ in their report of a chemical decaging strategy that can be used to specifically activate a POI (Fig. 1). The method relies on the use of a small-molecule palladium catalyst to remove an unsaturated protecting group (allyl or propargyl) from lysine. In recent years, application of transition metal-based catalysts in living biological systems has drawn great attention. Allyl and propargyl groups have superior bioorthogonality and unique reactivity to undergo tailored chemical transformations (for example, cleavage and cross-coupling) and so are ideal targets for selective catalysis by organometallic complexes7,8. In their report, Li et al.6 first screened a focused library of possible palladium catalysts, finding that propargyl carbamate (Proc) and, to a lesser extent, allyl carbamate could be effectively removed from lysine using biocompatible catalysts such as Pd(dba)2 and Allyl2Pd2Cl2. The authors validated the reaction on model compounds and purified proteins before turning to more
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
complex environments, in which Proc-lysine was site-specifically incorporated into a protein target using the genetically encoded unnatural amino acid insertion method9. To make optimal use of the caged lysine residue, Li et al.6 focused on proteins in which lysine residues are critical for activity, for example, nearby or at the active site of an enzyme. In this way, the wild-type activity of the caged protein would remain dormant until the palladium reagent–mediated Proc cleavage unleashed the free ε-amine of the lysine. As a case study, the authors expressed OspF, a phosphothreonine lyase, with Proclysine incorporated at a critical position (K134) such that its enzymatic activity was switched off. The subsequent addition of a membrane-permeable palladium reagent drove elimination of the Proc caging group, restoring OspF activity. With this tool, the authors confirmed that the selected lysine residue was essential for OspF activity and demonstrated that OspF altered the subcellular localization of its substrate, phosphorylated Erk (Fig. 1), explaining how OspF functions to impair cellular signaling pathways as part of bacterial infection. The method developed by Li et al.6 provides a small molecule–mediated chemical biology strategy for activating proteins by regenerating the nascent form of an embedded lysine residue. As the unnatural amino acid coding protocol for lysine replacement by Proc-lysine has been well established9, this new chemical rescue method can, in principle, be applied to any POI that contains a functionally important lysine. Such generality is important as currently there are few other general methods to discover small-molecule protein activators. Given the prevalence of lysine in regulating protein structure, stability, enzyme activity and protein-protein interactions, it is foreseeable that this lysine-dependent activation technique will find great use in elucidating the biological function of a protein target in a specific signaling pathway. In addition, this will be a great tool for studying catalytic roles of lysine residues in enzyme systems. The new strategy may also extend beyond lysines, as propargyl and allyl groups can be readily applied to protect the side chain of any amino acid that contains a nucleophilic atom, including lysine as well as tyrosine, serine, threonine, aspartate, glutamate and so on. However, further research will be needed to demonstrate whether the method can be expanded to these amino acids, as (i) the propargyl or allyl protecting group must be chemically stable within a certain timeframe, (ii) propargyl- or allyl-protected amino acids must be amenable to site-specific 329
news & views incorporation using either the genetic code expansion method or other protein engineering techniques10 and (iii) decaging by a cell-permeable biocompatible organometallic reagent must be rapid and efficient. Future investigations will prove (or disprove) these possibilities. ■
References
1. Schreiber, S.L. Nat. Chem. Biol. 1, 64–66 (2005). 2. Carlson, S.M. & White, F.M. ACS Chem. Biol. 6, 75–85 (2011). 3. Zorn, J.A. & Wells, J.A. Nat. Chem. Biol. 6, 179–188 (2010). 4. Bishop, A.C. & Chen, V.L. J. Chem. Biol. 2, 1–9 (2009). 5. Tarrant, M.K. & Cole, P.A. Annu. Rev. Biochem. 78, 797–825 (2009).
6. Li, J. et al. Nat. Chem. 6, 352–361 (2014). 7. Santra, M., Ko, S.K., Shin, I. & Ahn, K.H. Chem. Commun. (Camb.) 46, 3964–3966 (2010). 8. Garner, A.L., Song, F. & Koide, K. J. Am. Chem. Soc. 131, 5163–5171 (2009). 9. Liu, C.C. & Schultz, P.G. Annu. Rev. Biochem. 79, 413–444 (2010). 10. Muralidharan, V. & Muir, T.W. Nat. Methods 3, 429–438 (2006).
Competing financial interests
The authors declare no competing financial interests.
npg
© 2014 Nature America, Inc. All rights reserved.
Kun Qian and Yujun George Zheng are at the Department of Pharmaceutical and Biomedical
Sciences, University of Georgia, Athens, Georgia, USA. e-mail: [email protected]
330
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
