npg
© 2014 Nature America, Inc. All rights reserved.
news & views toward the carrier protein has limited the use of these halogenases in non-native contexts to evolutionary closely related systems as the protein-protein interactions that govern this specificity are still poorly understood. The need for carrier protein attachment of the substrate has also hitherto rendered NHI halogenases unsuitable for enzyme engineering. WelO5 is without this drawback, and furthermore it processes a compound substantially more complex than most of the known NHI halogenase substrates. Its potentially larger binding pocket will perhaps accommodate other substrates. However, though WeIO5 can process two distinctly different natural substrates, demonstrating a level of flexibility, it remains to be seen whether the wild-type enzyme can chlorinate or indeed brominate non-native substrates. Hillwig and Liu5 report that hapalindole J, a Fischerella muscicola metabolite with a
different ring system than the H. welwitschii alkaloids, is not chlorinated by WelO5, which suggests a certain degree of substrate specificity. This is certainly an aspect that will require further investigations into WelO5 and related enzymes. It will be exciting to see how far future studies will be able to push the development of halogenases for use in biocatalysis. Good candidates for the halogenation of electron-rich, activated carbon centers have already been identified in flavin- or vanadium-dependent halogenases; the discovery of WelO5, a radical halogenase, represents a potentially very useful tool for the halogenaton of unactivated alkyl groups. ■ Rebecca J.M. Goss and Sabine Grüschow are at the School of Chemistry, University of St. Andrews, St. Andrews, Fife, UK. e-mail: [email protected]
Published online 14 September 2014 doi:10.1038/nchembio.1649 References
1. Fowden, L. & Robinson, R. Proc. R. Soc. Lond. B Biol. Sci. 171, 5–18 (1968). 2. Gribble, G.W. Alkaloids Chem. Biol. 71, 1–165 (2012). 3. Smith, D.R.M., Grüschow, S. & Goss, R.J.M. Curr. Opin. Chem. Biol. 17, 276–283 (2013). 4. Butler, A. & Sandy, M. Nature 460, 848–854 (2009). 5. Hillwig, M.L. & Liu, X. Nat. Chem. Biol. doi:10.1038/ nchembio.1625 (14 September 2014) 6. Deng. H. & O’Hagan, D. Curr. Opin. Chem. Biol. 12, 582–592 (2008). 7. Krebs, C., Galonic Fujimori, D., Walsh, C.T. & Bollinger, J.M. Acc. Chem. Res. 40, 484–492 (2007). 8. Blasiak, L.C., Vaillancourt, F.H., Walsh, C.T. & Drennan, C.L. Nature 440, 368–371 (2006). 9. Gu, L. et al. Nature 459, 731–735 (2009). 10. Hillwig, M.L. et al. ChemBioChem 15, 665–669 (2014).
Competing financial interests
The authors declare no competing financial interests.
Protein homeostasis
Modeling UPR adaptive responses
Understanding the mechanisms that determine cell fate under endoplasmic reticulum (ER) stress had been hampered by the lack of models to study unfolded protein response (UPR) adaptive phases. The development of an engineered protein to conditionally induce its misfolding allowed the establishment of a resolvable ER stress condition.
Danilo B Medinas & Claudio Hetz
T
he accumulation of abnormally folded proteins in the ER lumen, a cellular condition referred to as ER stress, activates the UPR to restore proteostasis1. The UPR integrates information about the intensity and duration of the stimuli, engaging adaptive programs to reduce the unfolded protein load. Failure to overcome ER stress, however, engages the core apoptosis machinery2. Despite accumulated knowledge about UPR signaling mechanisms, the key elements regulating the transition from adaptive to proapoptotic phases of the UPR are not well understood. This information is essential to determine how cell fate is regulated under ER stress. This difficulty in the field is largely due to the lack of appropriate tools to study the discrete phases of the UPR separately. Classical pharmacological approaches cause irreversible ER damage that inexorably results in cell death, leading to paradoxical interpretations owing to the overlap of adaptive and proapoptotic signals1. In this issue, Raina and collaborators describe a
new genetic tool to cause acute ER stress that can be resolved by the cell3. The method developed results in robust and transient UPR activation without induction of apoptosis, opening the door to the discovery of new pathways supporting cell survival. Protein folding in the ER requires the concerted action of a network of chaperones and quality control mechanisms that ensure the generation of biologically active three-dimensional structures. Terminally misfolded proteins are retrotranslocated to the cytosol for degradation by the ubiquitinproteasome system. Physiological levels of ER stress are observed in most specialized secretory cells, where an increase in demand for protein synthesis and folding engages an adaptive and nonapoptotic UPR reaction that is essential to sustain cell function4. Chronic or irreversible ER stress is mostly observed in pathological conditions such as metabolic syndromes and neurodegenerative diseases5. The UPR is mediated by three types of stress sensors, including IRE1, PERK and ATF6. Each UPR signaling
nature chemical biology | VOL 10 | NOVEMBER 2014 | www.nature.com/naturechemicalbiology
branch has its own mode of action, kinetics of activation and regulatory mechanisms that depend on intrinsic features of the cell type affected, in addition to the source, intensity and duration of the stress stimuli1,6. The final outcome of the UPR reflects the capacity of the cell to reestablish proteostasis as well as the extent of cell damage caused by the injury1. Experimental approaches that enable the teasing apart of the adaptive and pro-apoptotic phases of the UPR are instrumental in the comprehension of molecular and cellular mechanisms governing the maintenance of proteostasis. This is especially relevant in physiological conditions where cells are functional with sustained levels of mild ER stress1,5. Raina and co-workers3 developed a strategy to induce adaptive ER stress while avoiding the irreversible damage to organelle homeostasis. The new method is based on the expression of an ER-localized HaloTag (ERHT) protein, termed ERHT, that can be destabilized upon addition of a small hydrophobic tag molecule to its 879
news & views Secretory signal
a Haloalkane reactive linker
Hydrophobic moiety
HA tag EGFP HaloTag
+
HyT36 binding site
HyT36
ER retention signal
ERHT
b
ERHTinducible misfolding
UPR adaptive genes
npg
© 2014 Nature America, Inc. All rights reserved.
ATF6 ATF6
Estrogen signaling
PERK IRE1α
PERK
Proteostasis
ATF6
HyT36
ATF6f
p-eIF2α ERHT
PERK
Misfolded ERHT
IRE1α
ER stress
IRE1α
Proteostasis restored
ATF4
XBP1s
Figure 1 | Inducible ERHT protein misfolding triggers resolvable ER stress. (a) Schematic representation of the ERHT protein destabilization by the small hydrophobic molecule HyT36. HA, hemagglutinin. (b) HyT36 causes misfolding of ERHT in cells, causing ER stress and induction of a transient adaptive UPR despite sustained accumulation of ERHT. Estrogen signaling was identified as a UPR downstream response during the recovery phase from ER stress.
surface7. The engineered construct is based on the fusion of the HaloTag dehalogenase bacterial system with GFP, together with the calreticulin ER translocation signal sequence and a classical ER retention sequence (Fig. 1a). They selected the HaloTag protein because it can form a covalent bond with a haloalkane-reactive linker, which the authors previously fused with hydrophobic moieties to induce the thermodynamic destabilization of proteins in the cytosol8. That study reported hydrophobic compounds bearing adamantyl groups that were nontoxic, cell permeable, highly stable and effective even in targeting specific proteins in vivo using zebrafish and a mouse
880
model of cancer8. Remarkably, the inducible, single-protein-misfolding ERHT approach was able to trigger activation of a robust UPR response with a similar magnitude as that caused by potent pharmacological ER stress agents3. Unexpectedly, pharmacological perturbations to ER proteostasis induced early apoptosis signals, whereas the misfolding of ERHT caused resolvable ER stress and transient UPR activation despite sustained expression of misfolded ERHT (Fig. 1b). This key observation allowed the global analysis of UPR adaptive responses using mRNA sequencing of cells during the recovery phase. Using this approach, the authors
uncovered a gene expression signature reflecting estrogen signaling. Upregulation of a cluster of genes downstream of the estrogen receptor was then validated in further experiments, even with classical ER stress agents. Inhibition of the IRE1 and XBP1 pathway abrogated UPR-induced activation of the estrogen pathway. Notably, a previous report demonstrated that XBP1 could bind and activate the transcription of the estrogen receptor9, providing a possible molecular explanation of how the UPR engages estrogen signaling. Recently, synergy between the UPR and estrogen signaling was also observed in human breast cancer10, underscoring the potential clinical implications of this prosurvival pathway. The use of ERHT to model adaptive ER stress responses will accelerate our understanding of how cells adjust the proteostasis network to enter into an adaptive state. Important applications will also emerge in defining the fine-tuning of the ‘UPRosome’ signaling scaffold1 and the identification of components determining cell fate decisions in physiology and disease. This study established a powerful platform to investigate prosurvival signals governed by the UPR. ■ Danilo B. Medinas and Claudio Hetz are at the Biomedical Neuroscience Institute, ICBM Faculty of Medicine, University of Chile, Santiago, Chile. Claudio Hetz is also at the Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA. e-mail: [email protected] References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Hetz, C. Nat. Rev. Mol. Cell Biol. 13, 89–102 (2012). Tabas, I. & Ron, D. Nat. Cell Biol. 13, 184–190 (2011). Raina, K. et al. Nat. Chem. Biol. 10, 957–962 (2014). Cornejo, V.H., Pihán, P., Vidal, R.L. & Hetz, C. IUBMB Life 65, 962–975 (2013). Rutkowski, D.T. & Hegde, R.S. J. Cell Biol. 189, 783–794 (2010). Walter, P. & Ron, D. Science 334, 1081–1086 (2011). Tae, H.S. et al. ChemBioChem 13, 538–541 (2012). Neklesa, T.K. et al. Nat. Chem. Biol. 7, 538–543 (2011). Ding, L. et al. Nucleic Acids Res. 31, 5266–5274 (2003). Cook, K.L. et al. FASEB J. doi:10.1096/fj.13-247353 (23 May 2014).
Competing financial interests
The authors declare no competing financial interests.
nature chemical biology | VOL 10 | NOVEMBER 2014 | www.nature.com/naturechemicalbiology
© 2014 Nature America, Inc. All rights reserved.
news & views toward the carrier protein has limited the use of these halogenases in non-native contexts to evolutionary closely related systems as the protein-protein interactions that govern this specificity are still poorly understood. The need for carrier protein attachment of the substrate has also hitherto rendered NHI halogenases unsuitable for enzyme engineering. WelO5 is without this drawback, and furthermore it processes a compound substantially more complex than most of the known NHI halogenase substrates. Its potentially larger binding pocket will perhaps accommodate other substrates. However, though WeIO5 can process two distinctly different natural substrates, demonstrating a level of flexibility, it remains to be seen whether the wild-type enzyme can chlorinate or indeed brominate non-native substrates. Hillwig and Liu5 report that hapalindole J, a Fischerella muscicola metabolite with a
different ring system than the H. welwitschii alkaloids, is not chlorinated by WelO5, which suggests a certain degree of substrate specificity. This is certainly an aspect that will require further investigations into WelO5 and related enzymes. It will be exciting to see how far future studies will be able to push the development of halogenases for use in biocatalysis. Good candidates for the halogenation of electron-rich, activated carbon centers have already been identified in flavin- or vanadium-dependent halogenases; the discovery of WelO5, a radical halogenase, represents a potentially very useful tool for the halogenaton of unactivated alkyl groups. ■ Rebecca J.M. Goss and Sabine Grüschow are at the School of Chemistry, University of St. Andrews, St. Andrews, Fife, UK. e-mail: [email protected]
Published online 14 September 2014 doi:10.1038/nchembio.1649 References
1. Fowden, L. & Robinson, R. Proc. R. Soc. Lond. B Biol. Sci. 171, 5–18 (1968). 2. Gribble, G.W. Alkaloids Chem. Biol. 71, 1–165 (2012). 3. Smith, D.R.M., Grüschow, S. & Goss, R.J.M. Curr. Opin. Chem. Biol. 17, 276–283 (2013). 4. Butler, A. & Sandy, M. Nature 460, 848–854 (2009). 5. Hillwig, M.L. & Liu, X. Nat. Chem. Biol. doi:10.1038/ nchembio.1625 (14 September 2014) 6. Deng. H. & O’Hagan, D. Curr. Opin. Chem. Biol. 12, 582–592 (2008). 7. Krebs, C., Galonic Fujimori, D., Walsh, C.T. & Bollinger, J.M. Acc. Chem. Res. 40, 484–492 (2007). 8. Blasiak, L.C., Vaillancourt, F.H., Walsh, C.T. & Drennan, C.L. Nature 440, 368–371 (2006). 9. Gu, L. et al. Nature 459, 731–735 (2009). 10. Hillwig, M.L. et al. ChemBioChem 15, 665–669 (2014).
Competing financial interests
The authors declare no competing financial interests.
Protein homeostasis
Modeling UPR adaptive responses
Understanding the mechanisms that determine cell fate under endoplasmic reticulum (ER) stress had been hampered by the lack of models to study unfolded protein response (UPR) adaptive phases. The development of an engineered protein to conditionally induce its misfolding allowed the establishment of a resolvable ER stress condition.
Danilo B Medinas & Claudio Hetz
T
he accumulation of abnormally folded proteins in the ER lumen, a cellular condition referred to as ER stress, activates the UPR to restore proteostasis1. The UPR integrates information about the intensity and duration of the stimuli, engaging adaptive programs to reduce the unfolded protein load. Failure to overcome ER stress, however, engages the core apoptosis machinery2. Despite accumulated knowledge about UPR signaling mechanisms, the key elements regulating the transition from adaptive to proapoptotic phases of the UPR are not well understood. This information is essential to determine how cell fate is regulated under ER stress. This difficulty in the field is largely due to the lack of appropriate tools to study the discrete phases of the UPR separately. Classical pharmacological approaches cause irreversible ER damage that inexorably results in cell death, leading to paradoxical interpretations owing to the overlap of adaptive and proapoptotic signals1. In this issue, Raina and collaborators describe a
new genetic tool to cause acute ER stress that can be resolved by the cell3. The method developed results in robust and transient UPR activation without induction of apoptosis, opening the door to the discovery of new pathways supporting cell survival. Protein folding in the ER requires the concerted action of a network of chaperones and quality control mechanisms that ensure the generation of biologically active three-dimensional structures. Terminally misfolded proteins are retrotranslocated to the cytosol for degradation by the ubiquitinproteasome system. Physiological levels of ER stress are observed in most specialized secretory cells, where an increase in demand for protein synthesis and folding engages an adaptive and nonapoptotic UPR reaction that is essential to sustain cell function4. Chronic or irreversible ER stress is mostly observed in pathological conditions such as metabolic syndromes and neurodegenerative diseases5. The UPR is mediated by three types of stress sensors, including IRE1, PERK and ATF6. Each UPR signaling
nature chemical biology | VOL 10 | NOVEMBER 2014 | www.nature.com/naturechemicalbiology
branch has its own mode of action, kinetics of activation and regulatory mechanisms that depend on intrinsic features of the cell type affected, in addition to the source, intensity and duration of the stress stimuli1,6. The final outcome of the UPR reflects the capacity of the cell to reestablish proteostasis as well as the extent of cell damage caused by the injury1. Experimental approaches that enable the teasing apart of the adaptive and pro-apoptotic phases of the UPR are instrumental in the comprehension of molecular and cellular mechanisms governing the maintenance of proteostasis. This is especially relevant in physiological conditions where cells are functional with sustained levels of mild ER stress1,5. Raina and co-workers3 developed a strategy to induce adaptive ER stress while avoiding the irreversible damage to organelle homeostasis. The new method is based on the expression of an ER-localized HaloTag (ERHT) protein, termed ERHT, that can be destabilized upon addition of a small hydrophobic tag molecule to its 879
news & views Secretory signal
a Haloalkane reactive linker
Hydrophobic moiety
HA tag EGFP HaloTag
+
HyT36 binding site
HyT36
ER retention signal
ERHT
b
ERHTinducible misfolding
UPR adaptive genes
npg
© 2014 Nature America, Inc. All rights reserved.
ATF6 ATF6
Estrogen signaling
PERK IRE1α
PERK
Proteostasis
ATF6
HyT36
ATF6f
p-eIF2α ERHT
PERK
Misfolded ERHT
IRE1α
ER stress
IRE1α
Proteostasis restored
ATF4
XBP1s
Figure 1 | Inducible ERHT protein misfolding triggers resolvable ER stress. (a) Schematic representation of the ERHT protein destabilization by the small hydrophobic molecule HyT36. HA, hemagglutinin. (b) HyT36 causes misfolding of ERHT in cells, causing ER stress and induction of a transient adaptive UPR despite sustained accumulation of ERHT. Estrogen signaling was identified as a UPR downstream response during the recovery phase from ER stress.
surface7. The engineered construct is based on the fusion of the HaloTag dehalogenase bacterial system with GFP, together with the calreticulin ER translocation signal sequence and a classical ER retention sequence (Fig. 1a). They selected the HaloTag protein because it can form a covalent bond with a haloalkane-reactive linker, which the authors previously fused with hydrophobic moieties to induce the thermodynamic destabilization of proteins in the cytosol8. That study reported hydrophobic compounds bearing adamantyl groups that were nontoxic, cell permeable, highly stable and effective even in targeting specific proteins in vivo using zebrafish and a mouse
880
model of cancer8. Remarkably, the inducible, single-protein-misfolding ERHT approach was able to trigger activation of a robust UPR response with a similar magnitude as that caused by potent pharmacological ER stress agents3. Unexpectedly, pharmacological perturbations to ER proteostasis induced early apoptosis signals, whereas the misfolding of ERHT caused resolvable ER stress and transient UPR activation despite sustained expression of misfolded ERHT (Fig. 1b). This key observation allowed the global analysis of UPR adaptive responses using mRNA sequencing of cells during the recovery phase. Using this approach, the authors
uncovered a gene expression signature reflecting estrogen signaling. Upregulation of a cluster of genes downstream of the estrogen receptor was then validated in further experiments, even with classical ER stress agents. Inhibition of the IRE1 and XBP1 pathway abrogated UPR-induced activation of the estrogen pathway. Notably, a previous report demonstrated that XBP1 could bind and activate the transcription of the estrogen receptor9, providing a possible molecular explanation of how the UPR engages estrogen signaling. Recently, synergy between the UPR and estrogen signaling was also observed in human breast cancer10, underscoring the potential clinical implications of this prosurvival pathway. The use of ERHT to model adaptive ER stress responses will accelerate our understanding of how cells adjust the proteostasis network to enter into an adaptive state. Important applications will also emerge in defining the fine-tuning of the ‘UPRosome’ signaling scaffold1 and the identification of components determining cell fate decisions in physiology and disease. This study established a powerful platform to investigate prosurvival signals governed by the UPR. ■ Danilo B. Medinas and Claudio Hetz are at the Biomedical Neuroscience Institute, ICBM Faculty of Medicine, University of Chile, Santiago, Chile. Claudio Hetz is also at the Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA. e-mail: [email protected] References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Hetz, C. Nat. Rev. Mol. Cell Biol. 13, 89–102 (2012). Tabas, I. & Ron, D. Nat. Cell Biol. 13, 184–190 (2011). Raina, K. et al. Nat. Chem. Biol. 10, 957–962 (2014). Cornejo, V.H., Pihán, P., Vidal, R.L. & Hetz, C. IUBMB Life 65, 962–975 (2013). Rutkowski, D.T. & Hegde, R.S. J. Cell Biol. 189, 783–794 (2010). Walter, P. & Ron, D. Science 334, 1081–1086 (2011). Tae, H.S. et al. ChemBioChem 13, 538–541 (2012). Neklesa, T.K. et al. Nat. Chem. Biol. 7, 538–543 (2011). Ding, L. et al. Nucleic Acids Res. 31, 5266–5274 (2003). Cook, K.L. et al. FASEB J. doi:10.1096/fj.13-247353 (23 May 2014).
Competing financial interests
The authors declare no competing financial interests.
nature chemical biology | VOL 10 | NOVEMBER 2014 | www.nature.com/naturechemicalbiology
