news & views
npg
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(ref. 9). This activation results in the PARylation of DNA-PKcs, which in turn leads to DNA-PKcs activation. A similar translocation occurs for TyrRS in response to RSV or cellular stress, including ER stress, metabolic stress (serum starvation) or heat shock, suggesting that nuclear translocation of TyrRS is a general PARP1-activating signal during stress3. Thus one function of tRNA synthetases appears to be activation of PARP1, and nuclear translocation is a potentially important component of this process. A next step is to determine how universal tRNA synthetase translocation is during stress and to identify the specific synthetases that are involved in this process10. It will also be important to determine whether
specific stressors result in the translocation of specific synthetases, and why. One possibility is that translocation of tRNA synthetase into the nucleus functions as a signal for abnormal translation in the cytoplasm. Thus PARP1 activation in response to this signal could provide an important link between protein proteostasis in the cytoplasm and activation of the appropriate nuclear stress response. ■ Florian J. Bock is at the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA, and Paul Chang is at the Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. e-mail: [email protected]
References
1. Sinclair, D.A. Annu. Rev. Pharmacol. Toxicol. 54, 363–380 (2014). 2. Kulkarni, S.S. & Cantó, C. Biochim. Biophys. Acta Basis Dis. doi:10.1016/j.bbadis.2014.10.005 (12 October 2014). 3. Sajish, M. & Schimmel, P. Nature doi:10.1038/nature14028 (22 December 2014). 4. Luo, X. & Kraus, W.L. Genes Dev. 26, 417–432 (2012). 5. Bai, P. & Cantó, C. Cell Metab. 16, 290–295 (2012). 6. Bai, P. et al. Cell Metab. 13, 461–468 (2011). 7. Scheibye-Knudsen, M. et al. Cell Metab. 20, 840–855 (2014). 8. Mouchiroud, L. et al. Cell 154, 430–441 (2013). 9. Sajish, M. et al. Nat. Chem. Biol. 8, 547–554 (2012). 10. Guo, M. & Schimmel, P. Nat. Chem. Biol. 9, 145–153 (2013).
Competing financial interests
The authors declare no competing financial interests.
METABOLISM
‘Channeling’ Hans Krebs
The physical arrangement of enzymes within native metabolic pathways is emerging as an important but underexplored area of molecular biology. Recent advances in mass spectrometry enabled confirmation of the proposal that the Krebs cycle enzymes form a complex and suggest that substrate channeling is the most likely benefit to this structural arrangement.
Danielle Tullman-Ercek
T
he Krebs cycle—a metabolic pathway in which acetate from biomolecules such as sugars and amino acids is used to generate energy for the cell—is essential to life, and its steps, down to the movement of electrons, have been memorized for decades by biochemistry students. Substrates of the cycle have varied and important roles within the cell (for example, oxaloacetate participates in other cell cycles and serves as an inhibitor to some pathway enzymes), and so the movement of substrates within this pathway, and control over substrates entering or exiting the cycle, are critical to the efficient function of the cell. For all that is known about the enzymes, substrates and products of the reactions that make up this cycle, however, the understanding of the spatial organization of the pathway is still in its infancy. In their recent report, Wu and Minteer provide the first experimental evidence that the enzymes of the Krebs cycle come together to form a defined complex within the mitochondria, and they use their results to inform a new model for the metabolic channeling of substrates among a subset of the enzymes1. Increasingly, scientists are discovering that critical biochemical pathways are 180
spatially organized within the cell2–5. Perhaps the most prominent example of the benefits of such organization is found in polyketide synthesis: the enzymes of these pathways form assembly lines that result in extraordinary efficiency and specificity for their target products6. However, a multitude of alternative scaffold arrangements also exist, from the encapsulated and tightly packed carbon-fixation enzymes within carboxysomes3 to the direct interactions of nitrogenase proteins7. It has long been proposed that the enzymes in the Krebs cycle similarly associate directly into a complex, or ‘metabolon’, and that this organization may greatly enhance pathway efficiencies. Indeed, clever work has made clear that such complexes are possible and would be advantageous, permitting the product of one reaction of the cycle to be channeled directly to the active site of the next reaction and thereby enhancing cycle efficiencies8. Yet experimental evidence for such a physical organization has been elusive, owing to the difficulties of analyzing the potentially transient and low-affinity interactions among the enzymes. Beyond informing our understanding of this classic and important pathway, an understanding of the organizational framework that exists
in nature would be of use to those wishing to design more efficient metabolic pathways for biochemical production and synthetic biology applications. Minteer and co-workers previously observed that crosslinking proteins in the mitochondrial lysate and depositing them on the anode of a pyruvate/air fuel cell led to significant enhancement in the performance of the cell as compared to one containing non-crosslinked samples9. This led to speculation that they could prepare a similarly crosslinked sample to search for and characterize resulting complexes via mass spectrometry. The newly published results are clear: the largest complex in their sample indeed appears to contain each of the eight Krebs cycle enzymes, and this complex disappears when the crosslinking is not performed, most likely because the interactions are relatively weak. The authors then set out to identify the crosslinks—which they hoped would be indicative of physiologically relevant interfaces—and use modeling to construct possible complex arrangements for the three most abundant enzymes (malate dehydrogenase, citrate synthase and aconitase, which also catalyze sequential reactions in the pathway) via a docking
nature chemical biology | VOL 11 | MARCH 2015 | www.nature.com/naturechemicalbiology
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b
c – +
–
+ + MDH
+
+ + + +
–
+ CS
Active sites
npg
© 2015 Nature America, Inc. All rights reserved.
–
+
+
ACON
+
Figure 1 | Crosslinking and mass spectrometry provide clues to substrate channeling among three Krebs cycle enzymes. (a) Crosslinks can form only between lysines (represented as the knobs and holes of a puzzle piece). Coupling crosslinking with mass spectrometry data yields clues informing the potential spatial organization of the various enzyme components of a complex (represented as puzzle pieces). (b) Surface electrostatic potentials reveal positively charged (purple) pathways connecting the active sites of the enzymes, along which intermediates may move. (c) A model of the channeling pathways in the Krebs cycle metabolon. Blue arrows show that the negatively charged substrates move from one active site to the next, perhaps along the positively charged channels. MDH, malate dehydrogenase; CS, citrate synthase; ACON, aconitase. Images courtesy of Christopher Jakobson (a,b) and Marilyn Slininger (c).
algorithm (Fig. 1a). They present two potential arrangements, along with models of the resulting electrostatics arrangements over the surfaces of the enzymes (Fig. 1b). Intriguingly, both arrangements resulted in the formation in positively charged channels connecting the three active sites—perfect for the shuttling of the negatively charged substrates between malate dehydrogenase, citrate synthase and aconitase (Fig. 1c). These results raise several exciting questions. First, what is the mechanism for a particular substrate to enter or exit the metabolon, and how is this regulated? The abundance of each substrate in a free state may be as important to control as their paths between the various active sites. Another question is whether the metabolon complex is transient—does a particular biochemical or ion disrupt the enzyme interactions? Are both of the potential arrangements they identified physiologically relevant, depending on conditions? Moreover, are all of the substrates channeled, or are only a subset of them shuttled between enzymes in this way? And, perhaps most importantly, does the complex form to
enable substrate channeling, or is there an alternative or additional purpose? This last question is of particular interest to those in metabolic engineering who wish to exploit the strategy—before it can be applied, the impact must be understood. Models and theoretical studies based on other arrangements of the complex predict that substrate channeling is possible and would lead to increased substrate transport rates, directly affecting flux through the pathway. However, it seems equally probable that some substrates in the cycle require channeling because of instability (for example, owing to the existence of an efficient side reaction) or to prevent or encourage bottlenecks at particular steps. To answer these questions and address the purpose of the formation of the complex, much more information about the metabolon structure—and any channels within it—will be needed. Tremendous advances in electron cryomicroscopy have resulted in improved resolution10 and make this technique one potential avenue to explore; while such structural determinations will not be trivial, the work by Wu and Minteer justifies and
nature chemical biology | VOL 11 | MARCH 2015 | www.nature.com/naturechemicalbiology
motivates such challenging but potentially transformative pursuits. ■ Danielle Tullman-Ercek is in the Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California, USA. e-mail: [email protected] References
1. Wu, F. & Minteer, S. Angew. Chem. Int. Edn. Engl. doi:10.1002/anie. 201409336 (23 December 2014). 2. Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W. & Davies, D.R. J. Biol. Chem. 263, 17857–17871 (1988). 3. Cameron, J.C., Wilson, S.C., Bernstein, S.L. & Kerfeld, C.A. Cell 155, 1131–1140 (2013). 4. Montero Llopis, P. et al. Nature 466, 77–81 (2010). 5. Savage, D.F., Afonso, B., Chen, A.H. & Silver, P.A. Science 327, 1258–1261 (2010). 6. Khosla, C., Herschlag, D., Cane, D.E. & Walsh, C.T. Biochemistry 53, 2875–2883 (2014). 7. Hu, Y. & Ribbe, M.W. Biochim. Biophys. Acta 1827, 1112–1122 (2013). 8. Ovádi, J. & Srere, P.A. in Microcompartmentation and Phase Separation in Cytoplasm: A Survey of Cell Biology (International Review of Cytology vol. 192) (eds. Walter, H., Brooks, D.E. & Srere, P.A.) 255–280 (Academic Press, 1999). 9. Moehlenbrock, M.J., Toby, T.K., Waheed, A. & Minteer, S.D. J. Am. Chem. Soc. 132, 6288–6289 (2010). 10. Bai, X.-C., McMullan, G. & Scheres, S.H.W. Trends Biochem. Sci. 40, 49–57 (2015).
Competing financial interests
The author declares no competing financial interests.
181
npg
© 2015 Nature America, Inc. All rights reserved.
(ref. 9). This activation results in the PARylation of DNA-PKcs, which in turn leads to DNA-PKcs activation. A similar translocation occurs for TyrRS in response to RSV or cellular stress, including ER stress, metabolic stress (serum starvation) or heat shock, suggesting that nuclear translocation of TyrRS is a general PARP1-activating signal during stress3. Thus one function of tRNA synthetases appears to be activation of PARP1, and nuclear translocation is a potentially important component of this process. A next step is to determine how universal tRNA synthetase translocation is during stress and to identify the specific synthetases that are involved in this process10. It will also be important to determine whether
specific stressors result in the translocation of specific synthetases, and why. One possibility is that translocation of tRNA synthetase into the nucleus functions as a signal for abnormal translation in the cytoplasm. Thus PARP1 activation in response to this signal could provide an important link between protein proteostasis in the cytoplasm and activation of the appropriate nuclear stress response. ■ Florian J. Bock is at the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA, and Paul Chang is at the Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. e-mail: [email protected]
References
1. Sinclair, D.A. Annu. Rev. Pharmacol. Toxicol. 54, 363–380 (2014). 2. Kulkarni, S.S. & Cantó, C. Biochim. Biophys. Acta Basis Dis. doi:10.1016/j.bbadis.2014.10.005 (12 October 2014). 3. Sajish, M. & Schimmel, P. Nature doi:10.1038/nature14028 (22 December 2014). 4. Luo, X. & Kraus, W.L. Genes Dev. 26, 417–432 (2012). 5. Bai, P. & Cantó, C. Cell Metab. 16, 290–295 (2012). 6. Bai, P. et al. Cell Metab. 13, 461–468 (2011). 7. Scheibye-Knudsen, M. et al. Cell Metab. 20, 840–855 (2014). 8. Mouchiroud, L. et al. Cell 154, 430–441 (2013). 9. Sajish, M. et al. Nat. Chem. Biol. 8, 547–554 (2012). 10. Guo, M. & Schimmel, P. Nat. Chem. Biol. 9, 145–153 (2013).
Competing financial interests
The authors declare no competing financial interests.
METABOLISM
‘Channeling’ Hans Krebs
The physical arrangement of enzymes within native metabolic pathways is emerging as an important but underexplored area of molecular biology. Recent advances in mass spectrometry enabled confirmation of the proposal that the Krebs cycle enzymes form a complex and suggest that substrate channeling is the most likely benefit to this structural arrangement.
Danielle Tullman-Ercek
T
he Krebs cycle—a metabolic pathway in which acetate from biomolecules such as sugars and amino acids is used to generate energy for the cell—is essential to life, and its steps, down to the movement of electrons, have been memorized for decades by biochemistry students. Substrates of the cycle have varied and important roles within the cell (for example, oxaloacetate participates in other cell cycles and serves as an inhibitor to some pathway enzymes), and so the movement of substrates within this pathway, and control over substrates entering or exiting the cycle, are critical to the efficient function of the cell. For all that is known about the enzymes, substrates and products of the reactions that make up this cycle, however, the understanding of the spatial organization of the pathway is still in its infancy. In their recent report, Wu and Minteer provide the first experimental evidence that the enzymes of the Krebs cycle come together to form a defined complex within the mitochondria, and they use their results to inform a new model for the metabolic channeling of substrates among a subset of the enzymes1. Increasingly, scientists are discovering that critical biochemical pathways are 180
spatially organized within the cell2–5. Perhaps the most prominent example of the benefits of such organization is found in polyketide synthesis: the enzymes of these pathways form assembly lines that result in extraordinary efficiency and specificity for their target products6. However, a multitude of alternative scaffold arrangements also exist, from the encapsulated and tightly packed carbon-fixation enzymes within carboxysomes3 to the direct interactions of nitrogenase proteins7. It has long been proposed that the enzymes in the Krebs cycle similarly associate directly into a complex, or ‘metabolon’, and that this organization may greatly enhance pathway efficiencies. Indeed, clever work has made clear that such complexes are possible and would be advantageous, permitting the product of one reaction of the cycle to be channeled directly to the active site of the next reaction and thereby enhancing cycle efficiencies8. Yet experimental evidence for such a physical organization has been elusive, owing to the difficulties of analyzing the potentially transient and low-affinity interactions among the enzymes. Beyond informing our understanding of this classic and important pathway, an understanding of the organizational framework that exists
in nature would be of use to those wishing to design more efficient metabolic pathways for biochemical production and synthetic biology applications. Minteer and co-workers previously observed that crosslinking proteins in the mitochondrial lysate and depositing them on the anode of a pyruvate/air fuel cell led to significant enhancement in the performance of the cell as compared to one containing non-crosslinked samples9. This led to speculation that they could prepare a similarly crosslinked sample to search for and characterize resulting complexes via mass spectrometry. The newly published results are clear: the largest complex in their sample indeed appears to contain each of the eight Krebs cycle enzymes, and this complex disappears when the crosslinking is not performed, most likely because the interactions are relatively weak. The authors then set out to identify the crosslinks—which they hoped would be indicative of physiologically relevant interfaces—and use modeling to construct possible complex arrangements for the three most abundant enzymes (malate dehydrogenase, citrate synthase and aconitase, which also catalyze sequential reactions in the pathway) via a docking
nature chemical biology | VOL 11 | MARCH 2015 | www.nature.com/naturechemicalbiology
news & views a
b
c – +
–
+ + MDH
+
+ + + +
–
+ CS
Active sites
npg
© 2015 Nature America, Inc. All rights reserved.
–
+
+
ACON
+
Figure 1 | Crosslinking and mass spectrometry provide clues to substrate channeling among three Krebs cycle enzymes. (a) Crosslinks can form only between lysines (represented as the knobs and holes of a puzzle piece). Coupling crosslinking with mass spectrometry data yields clues informing the potential spatial organization of the various enzyme components of a complex (represented as puzzle pieces). (b) Surface electrostatic potentials reveal positively charged (purple) pathways connecting the active sites of the enzymes, along which intermediates may move. (c) A model of the channeling pathways in the Krebs cycle metabolon. Blue arrows show that the negatively charged substrates move from one active site to the next, perhaps along the positively charged channels. MDH, malate dehydrogenase; CS, citrate synthase; ACON, aconitase. Images courtesy of Christopher Jakobson (a,b) and Marilyn Slininger (c).
algorithm (Fig. 1a). They present two potential arrangements, along with models of the resulting electrostatics arrangements over the surfaces of the enzymes (Fig. 1b). Intriguingly, both arrangements resulted in the formation in positively charged channels connecting the three active sites—perfect for the shuttling of the negatively charged substrates between malate dehydrogenase, citrate synthase and aconitase (Fig. 1c). These results raise several exciting questions. First, what is the mechanism for a particular substrate to enter or exit the metabolon, and how is this regulated? The abundance of each substrate in a free state may be as important to control as their paths between the various active sites. Another question is whether the metabolon complex is transient—does a particular biochemical or ion disrupt the enzyme interactions? Are both of the potential arrangements they identified physiologically relevant, depending on conditions? Moreover, are all of the substrates channeled, or are only a subset of them shuttled between enzymes in this way? And, perhaps most importantly, does the complex form to
enable substrate channeling, or is there an alternative or additional purpose? This last question is of particular interest to those in metabolic engineering who wish to exploit the strategy—before it can be applied, the impact must be understood. Models and theoretical studies based on other arrangements of the complex predict that substrate channeling is possible and would lead to increased substrate transport rates, directly affecting flux through the pathway. However, it seems equally probable that some substrates in the cycle require channeling because of instability (for example, owing to the existence of an efficient side reaction) or to prevent or encourage bottlenecks at particular steps. To answer these questions and address the purpose of the formation of the complex, much more information about the metabolon structure—and any channels within it—will be needed. Tremendous advances in electron cryomicroscopy have resulted in improved resolution10 and make this technique one potential avenue to explore; while such structural determinations will not be trivial, the work by Wu and Minteer justifies and
nature chemical biology | VOL 11 | MARCH 2015 | www.nature.com/naturechemicalbiology
motivates such challenging but potentially transformative pursuits. ■ Danielle Tullman-Ercek is in the Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, California, USA. e-mail: [email protected] References
1. Wu, F. & Minteer, S. Angew. Chem. Int. Edn. Engl. doi:10.1002/anie. 201409336 (23 December 2014). 2. Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W. & Davies, D.R. J. Biol. Chem. 263, 17857–17871 (1988). 3. Cameron, J.C., Wilson, S.C., Bernstein, S.L. & Kerfeld, C.A. Cell 155, 1131–1140 (2013). 4. Montero Llopis, P. et al. Nature 466, 77–81 (2010). 5. Savage, D.F., Afonso, B., Chen, A.H. & Silver, P.A. Science 327, 1258–1261 (2010). 6. Khosla, C., Herschlag, D., Cane, D.E. & Walsh, C.T. Biochemistry 53, 2875–2883 (2014). 7. Hu, Y. & Ribbe, M.W. Biochim. Biophys. Acta 1827, 1112–1122 (2013). 8. Ovádi, J. & Srere, P.A. in Microcompartmentation and Phase Separation in Cytoplasm: A Survey of Cell Biology (International Review of Cytology vol. 192) (eds. Walter, H., Brooks, D.E. & Srere, P.A.) 255–280 (Academic Press, 1999). 9. Moehlenbrock, M.J., Toby, T.K., Waheed, A. & Minteer, S.D. J. Am. Chem. Soc. 132, 6288–6289 (2010). 10. Bai, X.-C., McMullan, G. & Scheres, S.H.W. Trends Biochem. Sci. 40, 49–57 (2015).
Competing financial interests
The author declares no competing financial interests.
181
