npg
© 2015 Nature America, Inc. All rights reserved.
news and views following their surface nucleation (Fig. 1) and that the aggregates would then rightly be small enough to be the obligate oligomers. So naturally the prediction is that Brichos should shut down oligomer formation, and again that’s exactly what’s seen. But the pièce de résistance is the authors’ demonstration that mouse brain degradation caused by the introduction of Aβ42 is prevented by the addition of Brichos. An elegant application of chemical kinetics has made the full leap from molecule to malady. Naturally, those who favor other mechanistic approaches are unlikely to concede easily, but the smart money is on the next-generation questions, which are structural in nature. Three immediately come to mind. First, how does the homogeneous nucleation process work? Simplifying that issue, it has been recently shown that the primary nucleation event involves structural rearrangement and not association per se9. This leaves the well-studied though still-perplexing question of the refolding process at the core of understanding the nucleation step. Second, what are the critical features of the Aβ fibrils that permit the secondary process?
The Brichos finding, though not a direct answer, provides valuable clues, because it is now known that a partner can shut down this process. Of course the answer may be nuanced, because a 100–amino acid chaperone docked to a 42–amino acid peptide may well do more than simple competitive inhibition (i.e., even without binding to the secondary-nucleation site, it might obstruct that site once attached to its own binding site). In addition, the possibility that the secondary-nucleation site represents some type of ‘defect’ in the fibrillar surface cannot be ruled out. The third question is a bit more subtle but nonetheless just as important: why does the daughter fibril separate? And at what step does this happen—at the secondary nucleus or at some specific number of molecules assembled on a fibril surface? Moreover, what revoked the attractiveness of the surface of the parent fibril? Does the surface bind only to some transition state? Beyond these immediate questions are important issues relating to understanding the prominent polymorphism of the amyloids10. Polymorphism may not directly appear in the
form of the kinetic equations and may thus be quite a subtle contributor. Yet this issue is inevitable, given the premise that the nature of the fibril surface is an essential element to the mechanism. Of course, these structural issues have not been broached in the current work, nor need they have been. These are questions for future investigations, but the road to the future assuredly begins here. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Cohen, S.I. et al. Proc. Natl. Acad. Sci. USA 110, 9758–9763 (2013). 2. Cohen, S.I.A. et al. Nat. Struct. Mol. Biol. 22, 207–213 (2015). 3. Haass, C. & Selkoe, D.J. Nat. Rev. Mol. Cell Biol. 8, 101–112 (2007). 4. Kayed, R. et al. Science 300, 486–489 (2003). 5. Ferrone, F.A., Hofrichter, J. & Eaton, W.A. J. Mol. Biol. 183, 611–631 (1985). 6. Bishop, M.F. & Ferrone, F.A. Biophys. J. 46, 631–644 (1984). 7. Willander, H. et al. Proc. Natl. Acad. Sci. USA 109, 2325–2329 (2012). 8. Willander, H. et al. J. Biol. Chem. 287, 31608–31617 (2012). 9. Ferrone, F.A. J. Mol. Biol. 427, 287–290 (2015). 10. Tycko, R. Protein Sci. 23, 1528–1539 (2014).
The proteasome gets a grip on protein complexity Matthew A Humbard & Michael R Maurizi Amyloids escape elimination by the proteasome, and their accumulation and subsequent aggregation contribute to various neurodegenerative conditions. A signature feature of amyloidogenic proteins is extended sequences rich in single amino acids. In this issue, Matouschek and colleagues now show that, to initiate degradation, the proteasome prefers substrates that have disordered regions with complex amino acid composition, thus indicating why it fails to rid the cell of most amyloids. Polyubiquitination of a protein targets it for degradation by the proteasome, but the best proteasomal substrates also contain a disordered segment that contributes to the initiation of degradation1. This observation led Matouschek and colleagues to query how components of the proteasome that themselves have extended disordered regions, such as Rad35, manage to escape degradation2. To further address this issue, the authors conducted in vitro degradation assays, using proteins fused to various disordered domains, and surprisingly observed that the proteasome did not initiate degradation efficiently when the disordered regions were compositionally uniform, i.e., composed of multiple runs of single amino acids. These in vitro Matthew A. Humbard and Michael R. Maurizi are at the Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. e-mail: [email protected]
studies were corroborated by database interrogations that revealed a striking correlation between a protein’s metabolic half-life and the amino acid composition of a disordered region at either the N or C terminus of the protein. Thus, compositional bias within disordered regions, independently of the specific overrepresented amino acids, appears to fine-tune the selection of substrates by the proteasome in vivo3. The cellular proteome is in constant flux. Proteolysis is needed to maintain temporal control and proper balance in metabolic and regulatory pathways and to remove structurally or chemically damaged proteins. However, intracellular protein degradation must also be carefully regulated, and the proteasome, which is responsible for nearly all rapid protein degradation in the eukaryotic cytosol, is designed to be highly selective. The proteolytic sites of the proteasome are sequestered within the core particle in a chamber that can be reached only through narrow axial channels. Access is
nature structural & molecular biology volume 22 number 3 march 2015
further controlled by capping the axial channels with large regulatory particles composed of two subdomains: a lid complex with receptors that bind polyubiquitin attached to proteins marked for degradation, and a base complex, composed of ATP-dependent protein unfoldases, which unravels bound proteins and delivers them to the core particle through the axial channels4. This multicomponent structural paradigm is recapitulated in the Clp, Lon and FtsH families of ATP-dependent proteases found in bacteria and in the organelles of eukaryotic cells. All ATP-dependent proteases use a bipartite mode of substrate recognition and processing. To be degraded, proteins must contain a degradation signal (e.g., polyubiquitin in eukaryotic cells) that can be recognized by a specialized domain of the protease complex, and they must have an unstructured or flexible region that binds to a separate site within the degradation machinery. Indeed, earlier studies from the Matouschek laboratory1 showed that 181
news and views a
Ubn Ubn
4 ATP
4 ADP
b
Ubn
Ubn
npg
© 2015 Nature America, Inc. All rights reserved.
n ATP n ADP
Figure 1 Amino acid diversity influences substrate engagement by the proteasome. (a) Variegated sequences favor capture and degradation. Left, proteins conjugated to polyubiquitin (Ubn) bind to receptors in the proteasome lid complex (dashed, gray) and are then engaged by ATP-dependent protein unfoldases in the base complex (colored ellipsoids), which deliver the deubiquitinated protein to the degradation chamber in the core particle (dashed, beige). Binding to the base and translocation requires a disordered region (multicolored line). Right, upon ATP hydrolysis, mobile loops in the base undergo reversible displacement along the central axis (power strokes) to impart a translocating force to the polypeptide in the channel. Multiple site engagement might help coordinate the power strokes required to unfold the protein, thus allowing it to be translocated to the degradation chamber. (b) Repetitive sequences elude capture and degradation. Disordered regions with a uniform amino acid composition (brown and black line) form a limited number of productive contacts that may be insufficient to coordinate power strokes, thus leading to failed unfolding, backtracking and release.
disordered regions located at the N or C termini of proteins help initiate degradation; however, not all disordered regions support degradation to the same extent. By analyzing degradation of fusion proteins with C-terminal extensions corresponding to either disordered regions present in proteins known to be stable in vivo or proteins that had previously been shown to promote proteasomal degradation, Matouschek and colleagues3 demonstrated that disordered regions from metabolically stable proteins are unable to initiate degradation of the fusion proteins. Comparison of various chemical or physical characteristics of a set of 15 disordered regions revealed no correlation between degradation rates and properties such as total or net charge, hydrophobicity, helix propensity, volume or even disorder. However, remarkably, degradation rates correlated with the complexity of the disordered regions, which is a measure of each amino acid’s frequency in the disordered regions compared to its frequency in the global proteome. These data suggested that degradation 182
by the proteasome is influenced by the distribution of amino acids within the disordered regions. Proteins with disordered N- or C-terminal regions are common in higher eukaryotes. Unstructured polypeptide segments have evolved as functional domains in many proteins, but such regions can also arise as a result of mutations or environmental conditions that lead to clusters of tandem repeats within genes, alternative mRNA splicing that fuses intron sequences to exons or alternative translational start sites, and readthrough of translational stop codons that results in extensions to the canonical sequences. Matouschek and colleagues3 examined the in vivo degradation rates of 4,502 different mouse proteins, of which about 30% had either an N- or C-terminal disordered region. When the proteins were sorted according to compositional bias within the disordered regions, the median half-life of proteins with unbiased compositions was significantly shorter than the global median or the median for proteins with disordered regions with compositional
bias. The relationship was most striking for proteins with C-terminal disordered regions: the median half-life of proteins with compositional bias was 54 h compared to 41 h for proteins with unbiased compositions. This finding provides a plausible mechanism for the variation of protein degradation in various amyloid diseases. Stable variants of Huntingtin found in patients with Huntington’s disease contain 40–100 repeats of glutamine5. Matouschek and colleagues3 found that a synthetic variant with a polyQ stretch of 52 residues was a poor substrate for the proteasome, whereas a variant in which the polyQ segment was replaced by an unbiased sequence of equal length was degraded efficiently. Thus, the lack of sequence diversity in Huntingtin apparently underlies its ability to escape degradation by the proteasome. How is unbiased composition in a disordered region detected and used by the proteasome? A complete answer to this question requires an understanding of the number and nature of the interactions between polypeptides and the proteasomal translocation machinery and of the way force is exerted on the polypeptide chain to move it through the channel. Although the biased sequences examined by Matouschek and colleagues3 impeded degradation when located at the N or C termini of proteins, they did not hinder it when inserted internally, thus indicating that compositional bias acts at or before the initial protein-unfolding step. The authors propose that the proteasome might have a mechanism to recognize unbiased sequences, which they conjecture would entail multiple interaction sites with chemically different binding preferences (hydrophobic, charged, aromatic, etc.). A set of diverse interaction sites on the proteasome could accommodate substrates with a wide variety of complementary residues within unbiased regions. The sum of these interactions could provide sufficient binding affinity to retain the substrate for subsequent translocation. Another intriguing possibility is that the binding sites involved in the formation of the initiation complex are part of the translocation apparatus itself and that multiple different interactions are needed to coordinate translocation steps and to unfold the protein. Recent single-molecule studies6,7 have established that translocation and unfolding are both driven by similar power strokes that are delivered to polypeptides by mobile loops within the axial channel (Fig. 1). Unfolding requires that the power strokes be delivered in coordinated bursts in order to displace a sufficient length of the polypeptide to prevent the protein from collapsing back to a folded state. Conversely, translocation of an unfolded protein occurs rapidly and stochastically. Coordination implies allosteric
volume 22 number 3 march 2015 nature structural & molecular biology
news and views Regardless of the exact mechanism, the finding that polypeptides with unbiased compositions are preferential targets of the proteasome suggests that the substrate-recognition sites on the proteasome have evolved to process complex polypeptides at a cost of failure when encountering repetitive sequences. More complete knowledge of the number and nature of the peptide-binding sites within the translocation channels is crucial to further understand the mechanism of substrate selection by the proteasome and the determinants of protein half-lives in vivo. The dynamic character of the axial channel and the possible variability of binding specificities and kinetics during cycles of ATP hydrolysis8 make this a daunting problem. Six different gene products contribute to the hexameric unfoldase of the eukaryotic proteasome. It should be possible to exploit this diversity to construct hexamers
with variants of potential binding sites to help address how and where disordered regions are bound. Such insights might guide construction of proteasomes with altered specificity for the degradation of diseasecausing amyloids and other toxic proteins. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Inobe, T., Fishbain, S., Prakash, S. & Matouschek, A. Nat. Chem. Biol. 7, 161–167 (2011). 2. Fishbain, S., Prakash, S., Herrig, A., Elsasser, S. & Matouschek, A. Nat. Commun. 2, 192 (2011). 3. Fishbain, S. et al. Nat. Struct. Mol. Biol. 22, 214–221 (2015). 4. Matyskiela, M.E., Lander, G.C. & Martin, A. Nat. Struct. Mol. Biol. 20, 781–788 (2013). 5. Zuccato, C., Valenza, M. & Cattaneo, E. Physiol. Rev. 90, 905–981 (2010). 6. Aubin-Tam, M.E., Olivares, A.O., Sauer, R.T., Baker, T.A. & Lang, M.J. Cell 145, 257–267 (2011). 7. Sen, M. et al. Cell 155, 636–646 (2013). 8. Erales, J., Hoyt, M.A., Troll, F. & Coffino, P. J. Biol. Chem. 287, 18535–18543 (2012).
npg
© 2015 Nature America, Inc. All rights reserved.
communication among the ATPase subunits, which would in turn be influenced by allosteric effects of substrate binding on ATP hydrolysis rates. Unbiased composition of the disordered polypeptide might increase the probability of multiple contacts with variable sites within the channel, some of which are not allosteric and do not trigger ATP hydrolysis, thus allowing ATP to accumulate on several subunits before a burst that leads to unfolding. In this model, biased sequences can be translocated (as observed by Matouschek and colleagues) but do not have the allosteric properties to allow coordinated bursts of translocation and therefore do not permit unfolding. This mechanism would also explain why biased sequences located between stable folded domains of multidomain proteins lead to partial degradation and release of functional folded domains from the proteasome.
nature structural & molecular biology volume 22 number 3 march 2015
183
© 2015 Nature America, Inc. All rights reserved.
news and views following their surface nucleation (Fig. 1) and that the aggregates would then rightly be small enough to be the obligate oligomers. So naturally the prediction is that Brichos should shut down oligomer formation, and again that’s exactly what’s seen. But the pièce de résistance is the authors’ demonstration that mouse brain degradation caused by the introduction of Aβ42 is prevented by the addition of Brichos. An elegant application of chemical kinetics has made the full leap from molecule to malady. Naturally, those who favor other mechanistic approaches are unlikely to concede easily, but the smart money is on the next-generation questions, which are structural in nature. Three immediately come to mind. First, how does the homogeneous nucleation process work? Simplifying that issue, it has been recently shown that the primary nucleation event involves structural rearrangement and not association per se9. This leaves the well-studied though still-perplexing question of the refolding process at the core of understanding the nucleation step. Second, what are the critical features of the Aβ fibrils that permit the secondary process?
The Brichos finding, though not a direct answer, provides valuable clues, because it is now known that a partner can shut down this process. Of course the answer may be nuanced, because a 100–amino acid chaperone docked to a 42–amino acid peptide may well do more than simple competitive inhibition (i.e., even without binding to the secondary-nucleation site, it might obstruct that site once attached to its own binding site). In addition, the possibility that the secondary-nucleation site represents some type of ‘defect’ in the fibrillar surface cannot be ruled out. The third question is a bit more subtle but nonetheless just as important: why does the daughter fibril separate? And at what step does this happen—at the secondary nucleus or at some specific number of molecules assembled on a fibril surface? Moreover, what revoked the attractiveness of the surface of the parent fibril? Does the surface bind only to some transition state? Beyond these immediate questions are important issues relating to understanding the prominent polymorphism of the amyloids10. Polymorphism may not directly appear in the
form of the kinetic equations and may thus be quite a subtle contributor. Yet this issue is inevitable, given the premise that the nature of the fibril surface is an essential element to the mechanism. Of course, these structural issues have not been broached in the current work, nor need they have been. These are questions for future investigations, but the road to the future assuredly begins here. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Cohen, S.I. et al. Proc. Natl. Acad. Sci. USA 110, 9758–9763 (2013). 2. Cohen, S.I.A. et al. Nat. Struct. Mol. Biol. 22, 207–213 (2015). 3. Haass, C. & Selkoe, D.J. Nat. Rev. Mol. Cell Biol. 8, 101–112 (2007). 4. Kayed, R. et al. Science 300, 486–489 (2003). 5. Ferrone, F.A., Hofrichter, J. & Eaton, W.A. J. Mol. Biol. 183, 611–631 (1985). 6. Bishop, M.F. & Ferrone, F.A. Biophys. J. 46, 631–644 (1984). 7. Willander, H. et al. Proc. Natl. Acad. Sci. USA 109, 2325–2329 (2012). 8. Willander, H. et al. J. Biol. Chem. 287, 31608–31617 (2012). 9. Ferrone, F.A. J. Mol. Biol. 427, 287–290 (2015). 10. Tycko, R. Protein Sci. 23, 1528–1539 (2014).
The proteasome gets a grip on protein complexity Matthew A Humbard & Michael R Maurizi Amyloids escape elimination by the proteasome, and their accumulation and subsequent aggregation contribute to various neurodegenerative conditions. A signature feature of amyloidogenic proteins is extended sequences rich in single amino acids. In this issue, Matouschek and colleagues now show that, to initiate degradation, the proteasome prefers substrates that have disordered regions with complex amino acid composition, thus indicating why it fails to rid the cell of most amyloids. Polyubiquitination of a protein targets it for degradation by the proteasome, but the best proteasomal substrates also contain a disordered segment that contributes to the initiation of degradation1. This observation led Matouschek and colleagues to query how components of the proteasome that themselves have extended disordered regions, such as Rad35, manage to escape degradation2. To further address this issue, the authors conducted in vitro degradation assays, using proteins fused to various disordered domains, and surprisingly observed that the proteasome did not initiate degradation efficiently when the disordered regions were compositionally uniform, i.e., composed of multiple runs of single amino acids. These in vitro Matthew A. Humbard and Michael R. Maurizi are at the Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. e-mail: [email protected]
studies were corroborated by database interrogations that revealed a striking correlation between a protein’s metabolic half-life and the amino acid composition of a disordered region at either the N or C terminus of the protein. Thus, compositional bias within disordered regions, independently of the specific overrepresented amino acids, appears to fine-tune the selection of substrates by the proteasome in vivo3. The cellular proteome is in constant flux. Proteolysis is needed to maintain temporal control and proper balance in metabolic and regulatory pathways and to remove structurally or chemically damaged proteins. However, intracellular protein degradation must also be carefully regulated, and the proteasome, which is responsible for nearly all rapid protein degradation in the eukaryotic cytosol, is designed to be highly selective. The proteolytic sites of the proteasome are sequestered within the core particle in a chamber that can be reached only through narrow axial channels. Access is
nature structural & molecular biology volume 22 number 3 march 2015
further controlled by capping the axial channels with large regulatory particles composed of two subdomains: a lid complex with receptors that bind polyubiquitin attached to proteins marked for degradation, and a base complex, composed of ATP-dependent protein unfoldases, which unravels bound proteins and delivers them to the core particle through the axial channels4. This multicomponent structural paradigm is recapitulated in the Clp, Lon and FtsH families of ATP-dependent proteases found in bacteria and in the organelles of eukaryotic cells. All ATP-dependent proteases use a bipartite mode of substrate recognition and processing. To be degraded, proteins must contain a degradation signal (e.g., polyubiquitin in eukaryotic cells) that can be recognized by a specialized domain of the protease complex, and they must have an unstructured or flexible region that binds to a separate site within the degradation machinery. Indeed, earlier studies from the Matouschek laboratory1 showed that 181
news and views a
Ubn Ubn
4 ATP
4 ADP
b
Ubn
Ubn
npg
© 2015 Nature America, Inc. All rights reserved.
n ATP n ADP
Figure 1 Amino acid diversity influences substrate engagement by the proteasome. (a) Variegated sequences favor capture and degradation. Left, proteins conjugated to polyubiquitin (Ubn) bind to receptors in the proteasome lid complex (dashed, gray) and are then engaged by ATP-dependent protein unfoldases in the base complex (colored ellipsoids), which deliver the deubiquitinated protein to the degradation chamber in the core particle (dashed, beige). Binding to the base and translocation requires a disordered region (multicolored line). Right, upon ATP hydrolysis, mobile loops in the base undergo reversible displacement along the central axis (power strokes) to impart a translocating force to the polypeptide in the channel. Multiple site engagement might help coordinate the power strokes required to unfold the protein, thus allowing it to be translocated to the degradation chamber. (b) Repetitive sequences elude capture and degradation. Disordered regions with a uniform amino acid composition (brown and black line) form a limited number of productive contacts that may be insufficient to coordinate power strokes, thus leading to failed unfolding, backtracking and release.
disordered regions located at the N or C termini of proteins help initiate degradation; however, not all disordered regions support degradation to the same extent. By analyzing degradation of fusion proteins with C-terminal extensions corresponding to either disordered regions present in proteins known to be stable in vivo or proteins that had previously been shown to promote proteasomal degradation, Matouschek and colleagues3 demonstrated that disordered regions from metabolically stable proteins are unable to initiate degradation of the fusion proteins. Comparison of various chemical or physical characteristics of a set of 15 disordered regions revealed no correlation between degradation rates and properties such as total or net charge, hydrophobicity, helix propensity, volume or even disorder. However, remarkably, degradation rates correlated with the complexity of the disordered regions, which is a measure of each amino acid’s frequency in the disordered regions compared to its frequency in the global proteome. These data suggested that degradation 182
by the proteasome is influenced by the distribution of amino acids within the disordered regions. Proteins with disordered N- or C-terminal regions are common in higher eukaryotes. Unstructured polypeptide segments have evolved as functional domains in many proteins, but such regions can also arise as a result of mutations or environmental conditions that lead to clusters of tandem repeats within genes, alternative mRNA splicing that fuses intron sequences to exons or alternative translational start sites, and readthrough of translational stop codons that results in extensions to the canonical sequences. Matouschek and colleagues3 examined the in vivo degradation rates of 4,502 different mouse proteins, of which about 30% had either an N- or C-terminal disordered region. When the proteins were sorted according to compositional bias within the disordered regions, the median half-life of proteins with unbiased compositions was significantly shorter than the global median or the median for proteins with disordered regions with compositional
bias. The relationship was most striking for proteins with C-terminal disordered regions: the median half-life of proteins with compositional bias was 54 h compared to 41 h for proteins with unbiased compositions. This finding provides a plausible mechanism for the variation of protein degradation in various amyloid diseases. Stable variants of Huntingtin found in patients with Huntington’s disease contain 40–100 repeats of glutamine5. Matouschek and colleagues3 found that a synthetic variant with a polyQ stretch of 52 residues was a poor substrate for the proteasome, whereas a variant in which the polyQ segment was replaced by an unbiased sequence of equal length was degraded efficiently. Thus, the lack of sequence diversity in Huntingtin apparently underlies its ability to escape degradation by the proteasome. How is unbiased composition in a disordered region detected and used by the proteasome? A complete answer to this question requires an understanding of the number and nature of the interactions between polypeptides and the proteasomal translocation machinery and of the way force is exerted on the polypeptide chain to move it through the channel. Although the biased sequences examined by Matouschek and colleagues3 impeded degradation when located at the N or C termini of proteins, they did not hinder it when inserted internally, thus indicating that compositional bias acts at or before the initial protein-unfolding step. The authors propose that the proteasome might have a mechanism to recognize unbiased sequences, which they conjecture would entail multiple interaction sites with chemically different binding preferences (hydrophobic, charged, aromatic, etc.). A set of diverse interaction sites on the proteasome could accommodate substrates with a wide variety of complementary residues within unbiased regions. The sum of these interactions could provide sufficient binding affinity to retain the substrate for subsequent translocation. Another intriguing possibility is that the binding sites involved in the formation of the initiation complex are part of the translocation apparatus itself and that multiple different interactions are needed to coordinate translocation steps and to unfold the protein. Recent single-molecule studies6,7 have established that translocation and unfolding are both driven by similar power strokes that are delivered to polypeptides by mobile loops within the axial channel (Fig. 1). Unfolding requires that the power strokes be delivered in coordinated bursts in order to displace a sufficient length of the polypeptide to prevent the protein from collapsing back to a folded state. Conversely, translocation of an unfolded protein occurs rapidly and stochastically. Coordination implies allosteric
volume 22 number 3 march 2015 nature structural & molecular biology
news and views Regardless of the exact mechanism, the finding that polypeptides with unbiased compositions are preferential targets of the proteasome suggests that the substrate-recognition sites on the proteasome have evolved to process complex polypeptides at a cost of failure when encountering repetitive sequences. More complete knowledge of the number and nature of the peptide-binding sites within the translocation channels is crucial to further understand the mechanism of substrate selection by the proteasome and the determinants of protein half-lives in vivo. The dynamic character of the axial channel and the possible variability of binding specificities and kinetics during cycles of ATP hydrolysis8 make this a daunting problem. Six different gene products contribute to the hexameric unfoldase of the eukaryotic proteasome. It should be possible to exploit this diversity to construct hexamers
with variants of potential binding sites to help address how and where disordered regions are bound. Such insights might guide construction of proteasomes with altered specificity for the degradation of diseasecausing amyloids and other toxic proteins. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Inobe, T., Fishbain, S., Prakash, S. & Matouschek, A. Nat. Chem. Biol. 7, 161–167 (2011). 2. Fishbain, S., Prakash, S., Herrig, A., Elsasser, S. & Matouschek, A. Nat. Commun. 2, 192 (2011). 3. Fishbain, S. et al. Nat. Struct. Mol. Biol. 22, 214–221 (2015). 4. Matyskiela, M.E., Lander, G.C. & Martin, A. Nat. Struct. Mol. Biol. 20, 781–788 (2013). 5. Zuccato, C., Valenza, M. & Cattaneo, E. Physiol. Rev. 90, 905–981 (2010). 6. Aubin-Tam, M.E., Olivares, A.O., Sauer, R.T., Baker, T.A. & Lang, M.J. Cell 145, 257–267 (2011). 7. Sen, M. et al. Cell 155, 636–646 (2013). 8. Erales, J., Hoyt, M.A., Troll, F. & Coffino, P. J. Biol. Chem. 287, 18535–18543 (2012).
npg
© 2015 Nature America, Inc. All rights reserved.
communication among the ATPase subunits, which would in turn be influenced by allosteric effects of substrate binding on ATP hydrolysis rates. Unbiased composition of the disordered polypeptide might increase the probability of multiple contacts with variable sites within the channel, some of which are not allosteric and do not trigger ATP hydrolysis, thus allowing ATP to accumulate on several subunits before a burst that leads to unfolding. In this model, biased sequences can be translocated (as observed by Matouschek and colleagues) but do not have the allosteric properties to allow coordinated bursts of translocation and therefore do not permit unfolding. This mechanism would also explain why biased sequences located between stable folded domains of multidomain proteins lead to partial degradation and release of functional folded domains from the proteasome.
nature structural & molecular biology volume 22 number 3 march 2015
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