2023
Robb, Christina Glen; Dao, Thuy P.; Ujma, Jakub; Castañeda, Carlos A.; Beveridge, Rebecca
In: J. Am. Chem. Soc., 2023, ISSN: 1520-5126.
@article{Robb2023,
title = {Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation},
author = {Christina Glen Robb and Thuy P. Dao and Jakub Ujma and Carlos A. Castañeda and Rebecca Beveridge},
doi = {10.1021/jacs.3c00756},
issn = {1520-5126},
year = {2023},
date = {2023-06-05},
journal = {J. Am. Chem. Soc.},
publisher = {American Chemical Society (ACS)},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Dao, Thuy P; Castañeda, Carlos A
We con-dense if we want to; We can’t leave AZUL outside Journal Article
In: Structure, vol. 31, no. 4, pp. 369–371, 2023, ISSN: 1878-4186.
@article{pmid37028393,
title = {We con-dense if we want to; We can't leave AZUL outside},
author = {Thuy P Dao and Carlos A Castañeda},
doi = {10.1016/j.str.2023.03.006},
issn = {1878-4186},
year = {2023},
date = {2023-04-01},
journal = {Structure},
volume = {31},
number = {4},
pages = {369--371},
abstract = {In this issue of Structure, Buel et al. (2023) combined NMR data with AlphaFold2 to map out the interaction between the AZUL domain of ubiquitin ligase E6AP and UBQLN1/2 UBA. The authors demonstrated that this interaction enhances the self-association of the helix neighboring UBA and enables E6AP to localize to UBQLN2 droplets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this issue of Structure, Buel et al. (2023) combined NMR data with AlphaFold2 to map out the interaction between the AZUL domain of ubiquitin ligase E6AP and UBQLN1/2 UBA. The authors demonstrated that this interaction enhances the self-association of the helix neighboring UBA and enables E6AP to localize to UBQLN2 droplets.
Galagedera, Sarasi K K; Dao, Thuy P; Enos, Suzanne E; Chaudhuri, Antara; Schmit, Jeremy D; Castañeda, Carlos A
Decoding optimal ligand design for multicomponent condensates
2023.
@{pmid36993708,
title = {Decoding optimal ligand design for multicomponent condensates},
author = {Sarasi K K Galagedera and Thuy P Dao and Suzanne E Enos and Antara Chaudhuri and Jeremy D Schmit and Carlos A Castañeda},
doi = {10.1101/2023.03.13.532222},
year = {2023},
date = {2023-03-01},
journal = {bioRxiv},
abstract = {Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub's ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
HIGHLIGHTS: There is an optimal polyUb ligand architecture/design that promotes multicomponent phase separation, as polyUb hubs whose Ub units are too close together or too far apart are not effective drivers of phase separation for either UBQLN2 450-624 or full-length UBQLN2.Theoretical modeling reveals that Ub incurs a significant inclusion energetic penalty that is balanced by polyUb's ability to act as a hub to amplify UBQLN2-UBQLN2 interactions that facilitate phase separation.Naturally-occurring M1-linked polyUb chains are optimized to maximize phase separation with UBQLN2.Different linkages used in the Ub code deliver biochemical information via Ub-Ub spacing, whereby different outcomes are regulated by the emergent properties of Ub-containing biomolecular condensates.
SIGNIFICANCE: Biomolecular condensates are essential for cellular processes and are linked to human diseases when dysregulated. These condensates are hypothesized to assemble via phase transitions of a few key driver macromolecules and are further modulated by the interactions with ligands. Previous work showed that monovalent ligands inhibit driver phase transitions whereas multivalent ligand hubs comprising several identical binding sites to drivers promote phase transitions. Here, using a library of designed ligand hubs with decreasing or increasing spacings between binding sites and altered binding affinities with drivers, we employ theory and experiments to establish a new set of rules that govern how ligand hubs affect driver phase transitions. Our findings reveal that effects of macromolecules can be manipulated through emergent properties.},
keywords = {},
pubstate = {published},
tppubtype = {}
}
Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub’s ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
HIGHLIGHTS: There is an optimal polyUb ligand architecture/design that promotes multicomponent phase separation, as polyUb hubs whose Ub units are too close together or too far apart are not effective drivers of phase separation for either UBQLN2 450-624 or full-length UBQLN2.Theoretical modeling reveals that Ub incurs a significant inclusion energetic penalty that is balanced by polyUb’s ability to act as a hub to amplify UBQLN2-UBQLN2 interactions that facilitate phase separation.Naturally-occurring M1-linked polyUb chains are optimized to maximize phase separation with UBQLN2.Different linkages used in the Ub code deliver biochemical information via Ub-Ub spacing, whereby different outcomes are regulated by the emergent properties of Ub-containing biomolecular condensates.
SIGNIFICANCE: Biomolecular condensates are essential for cellular processes and are linked to human diseases when dysregulated. These condensates are hypothesized to assemble via phase transitions of a few key driver macromolecules and are further modulated by the interactions with ligands. Previous work showed that monovalent ligands inhibit driver phase transitions whereas multivalent ligand hubs comprising several identical binding sites to drivers promote phase transitions. Here, using a library of designed ligand hubs with decreasing or increasing spacings between binding sites and altered binding affinities with drivers, we employ theory and experiments to establish a new set of rules that govern how ligand hubs affect driver phase transitions. Our findings reveal that effects of macromolecules can be manipulated through emergent properties.
HIGHLIGHTS: There is an optimal polyUb ligand architecture/design that promotes multicomponent phase separation, as polyUb hubs whose Ub units are too close together or too far apart are not effective drivers of phase separation for either UBQLN2 450-624 or full-length UBQLN2.Theoretical modeling reveals that Ub incurs a significant inclusion energetic penalty that is balanced by polyUb’s ability to act as a hub to amplify UBQLN2-UBQLN2 interactions that facilitate phase separation.Naturally-occurring M1-linked polyUb chains are optimized to maximize phase separation with UBQLN2.Different linkages used in the Ub code deliver biochemical information via Ub-Ub spacing, whereby different outcomes are regulated by the emergent properties of Ub-containing biomolecular condensates.
SIGNIFICANCE: Biomolecular condensates are essential for cellular processes and are linked to human diseases when dysregulated. These condensates are hypothesized to assemble via phase transitions of a few key driver macromolecules and are further modulated by the interactions with ligands. Previous work showed that monovalent ligands inhibit driver phase transitions whereas multivalent ligand hubs comprising several identical binding sites to drivers promote phase transitions. Here, using a library of designed ligand hubs with decreasing or increasing spacings between binding sites and altered binding affinities with drivers, we employ theory and experiments to establish a new set of rules that govern how ligand hubs affect driver phase transitions. Our findings reveal that effects of macromolecules can be manipulated through emergent properties.
Raymond-Smiedy, Peter; Bucknor, Barrington; Yang, Yiran; Zheng, Tongyin; Castañeda, Carlos A
A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro Journal Article
In: Methods Mol Biol, vol. 2551, pp. 515–541, 2023, ISSN: 1940-6029.
@article{pmid36310223,
title = {A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro},
author = {Peter Raymond-Smiedy and Barrington Bucknor and Yiran Yang and Tongyin Zheng and Carlos A Castañeda},
doi = {10.1007/978-1-0716-2597-2_32},
issn = {1940-6029},
year = {2023},
date = {2023-01-01},
journal = {Methods Mol Biol},
volume = {2551},
pages = {515--541},
abstract = {Liquid-liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature-concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Liquid-liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature-concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.
2022
Dao, Thuy P; Yang, Yiran; Presti, Maria F; Cosgrove, Michael S; Hopkins, Jesse B; Ma, Weikang; Loh, Stewart N; Castañeda, Carlos A
Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains Journal Article
In: EMBO Rep, vol. 23, no. 8, pp. e55056, 2022, ISSN: 1469-3178.
@article{pmid35762418,
title = {Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains},
author = {Thuy P Dao and Yiran Yang and Maria F Presti and Michael S Cosgrove and Jesse B Hopkins and Weikang Ma and Stewart N Loh and Carlos A Castañeda},
doi = {10.15252/embr.202255056},
issn = {1469-3178},
year = {2022},
date = {2022-08-01},
journal = {EMBO Rep},
volume = {23},
number = {8},
pages = {e55056},
abstract = {Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.
2021
Riley, Julia F; Fioramonti, Peter J; Rusnock, Amber K; Hehnly, Heidi; Castañeda, Carlos A
ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells Journal Article
In: J Neurochem, vol. 159, no. 1, pp. 145–155, 2021, ISSN: 1471-4159.
@article{pmid34129687,
title = {ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells},
author = {Julia F Riley and Peter J Fioramonti and Amber K Rusnock and Heidi Hehnly and Carlos A Castañeda},
doi = {10.1111/jnc.15453},
issn = {1471-4159},
year = {2021},
date = {2021-10-01},
journal = {J Neurochem},
volume = {159},
number = {1},
pages = {145--155},
abstract = {Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.
Zheng, Tongyin; Galagedera, Sarasi K K; Castañeda, Carlos A
In: Protein Sci, vol. 30, no. 7, pp. 1467–1481, 2021, ISSN: 1469-896X.
@article{pmid34029402,
title = {Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation},
author = {Tongyin Zheng and Sarasi K K Galagedera and Carlos A Castañeda},
doi = {10.1002/pro.4128},
issn = {1469-896X},
year = {2021},
date = {2021-07-01},
journal = {Protein Sci},
volume = {30},
number = {7},
pages = {1467--1481},
abstract = {Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are low-complexity STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1-I and residues 555-570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N-terminal ubiquitin-like (UBL) and C-terminal ubiquitin-associated (UBA) domains, respectively. Between these two folded domains are low-complexity STI1-I and STI1-II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1-II region enables UBQLN2 to undergo liquid-liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1-I and residues 555-570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.
Namitz, Kevin E W; Zheng, Tongyin; Canning, Ashley J; Alicea-Velazquez, Nilda L; Castañeda, Carlos A; Cosgrove, Michael S; Hanes, Steven D
Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism Journal Article
In: Commun Biol, vol. 4, no. 1, pp. 398, 2021, ISSN: 2399-3642.
@article{pmid33767358,
title = {Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism},
author = {Kevin E W Namitz and Tongyin Zheng and Ashley J Canning and Nilda L Alicea-Velazquez and Carlos A Castañeda and Michael S Cosgrove and Steven D Hanes},
doi = {10.1038/s42003-021-01906-8},
issn = {2399-3642},
year = {2021},
date = {2021-03-01},
journal = {Commun Biol},
volume = {4},
number = {1},
pages = {398},
abstract = {Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the "CTD code" that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS) of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4-5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS) of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4-5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.
2020
Dao, Thuy P; Castañeda, Carlos A
Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation? Journal Article
In: Bioessays, vol. 42, no. 11, pp. e2000036, 2020, ISSN: 1521-1878.
@article{pmid32881044,
title = {Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?},
author = {Thuy P Dao and Carlos A Castañeda},
doi = {10.1002/bies.202000036},
issn = {1521-1878},
year = {2020},
date = {2020-11-01},
journal = {Bioessays},
volume = {42},
number = {11},
pages = {e2000036},
abstract = {Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.
Zheng, Tongyin; Yang, Yiran; Castañeda, Carlos A
Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery Journal Article
In: Biochem J, vol. 477, no. 18, pp. 3471–3497, 2020, ISSN: 1470-8728.
@article{pmid32965492,
title = {Structure, dynamics and functions of UBQLNs: at the crossroads of protein quality control machinery},
author = {Tongyin Zheng and Yiran Yang and Carlos A Castañeda},
doi = {10.1042/BCJ20190497},
issn = {1470-8728},
year = {2020},
date = {2020-09-01},
journal = {Biochem J},
volume = {477},
number = {18},
pages = {3471--3497},
abstract = {Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.
2019
Cable, Jennifer; Brangwynne, Clifford; Seydoux, Geraldine; Cowburn, David; Pappu, Rohit V; Castañeda, Carlos A; Berchowitz, Luke E; Chen, Zhijuan; Jonikas, Martin; Dernburg, Abby; Mittag, Tanja; Fawzi, Nicolas L
Phase separation in biology and disease-a symposium report Journal Article
In: Ann N Y Acad Sci, vol. 1452, no. 1, pp. 3–11, 2019, ISSN: 1749-6632.
@article{pmid31199001,
title = {Phase separation in biology and disease-a symposium report},
author = {Jennifer Cable and Clifford Brangwynne and Geraldine Seydoux and David Cowburn and Rohit V Pappu and Carlos A Castañeda and Luke E Berchowitz and Zhijuan Chen and Martin Jonikas and Abby Dernburg and Tanja Mittag and Nicolas L Fawzi},
doi = {10.1111/nyas.14126},
issn = {1749-6632},
year = {2019},
date = {2019-09-01},
journal = {Ann N Y Acad Sci},
volume = {1452},
number = {1},
pages = {3--11},
abstract = {Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington's disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Phase separation of multivalent protein and RNA molecules enables cells the formation of reversible nonstoichiometric, membraneless assemblies. These assemblies, referred to as biomolecular condensates, help with the spatial organization and compartmentalization of cellular matter. Each biomolecular condensate is defined by a distinct macromolecular composition. Distinct condensates have distinct preferential locations within cells, and they are associated with distinct biological functions, including DNA replication, RNA metabolism, signal transduction, synaptic transmission, and stress response. Several proteins found in biomolecular condensates have also been implicated in disease, including Huntington’s disease, amyotrophic lateral sclerosis, and several types of cancer. Disease-associated mutations in these proteins have been found to affect the material properties of condensates as well as the driving forces for phase separation. Understanding the intrinsic and extrinsic forces driving the formation and dissolution of biomolecular condensates via spontaneous and driven phase separation is an important step in understanding the processes associated with biological regulation in health and disease.
Dao, Thuy P; Martyniak, Brian; Canning, Ashley J; Lei, Yongna; Colicino, Erica G; Cosgrove, Michael S; Hehnly, Heidi; Castañeda, Carlos A
ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner Journal Article
In: Structure, vol. 27, no. 6, pp. 937–951.e5, 2019, ISSN: 1878-4186.
@article{pmid30982635,
title = {ALS-Linked Mutations Affect UBQLN2 Oligomerization and Phase Separation in a Position- and Amino Acid-Dependent Manner},
author = {Thuy P Dao and Brian Martyniak and Ashley J Canning and Yongna Lei and Erica G Colicino and Michael S Cosgrove and Heidi Hehnly and Carlos A Castañeda},
doi = {10.1016/j.str.2019.03.012},
issn = {1878-4186},
year = {2019},
date = {2019-06-01},
journal = {Structure},
volume = {27},
number = {6},
pages = {937--951.e5},
abstract = {Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.
Yang, Yiran; Jones, Holly B; Dao, Thuy P; Castañeda, Carlos A
Single Amino Acid Substitutions in Stickers, but Not Spacers, Substantially Alter UBQLN2 Phase Transitions and Dense Phase Material Properties Journal Article
In: J Phys Chem B, vol. 123, no. 17, pp. 3618–3629, 2019, ISSN: 1520-5207.
@article{pmid30925840,
title = {Single Amino Acid Substitutions in Stickers, but Not Spacers, Substantially Alter UBQLN2 Phase Transitions and Dense Phase Material Properties},
author = {Yiran Yang and Holly B Jones and Thuy P Dao and Carlos A Castañeda},
doi = {10.1021/acs.jpcb.9b01024},
issn = {1520-5207},
year = {2019},
date = {2019-05-01},
journal = {J Phys Chem B},
volume = {123},
number = {17},
pages = {3618--3629},
abstract = {UBQLN2 450-624 oligomerizes and undergoes temperature-responsive liquid-liquid phase transitions following a closed-loop temperature-concentration phase diagram. We recently showed that disease-linked mutations to UBQLN2 450-624 impart highly varying effects to its phase behavior, ranging from little change to significant decrease of saturation concentration and formation of gels and aggregates. However, how single mutations lead to these properties is unknown. Here, we use UBQLN2 450-624 as a model system to study the sequence determinants of phase separation. We hypothesized that UBQLN2 450-624 regions previously identified to promote its oligomerization are the "stickers" that drive interchain interactions and phase separation. We systematically investigated how phase behavior is affected by all 19 possible single amino acid substitutions at three sticker and two "spacer" (sequences separating stickers) positions. Overall, substitutions to stickers, but not spacers, substantially altered the shape of the phase diagram. Within the sticker regions, increasing hydrophobicity decreased saturation concentrations at low temperatures and enhanced oligomerization propensity and viscoelasticity of the dense phase. Conversely, substitutions to acidic residues at all positions greatly increased saturation concentrations. Our data demonstrate that single amino acid substitutions follow a molecular code to tune phase transition behavior of biopolymers.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
UBQLN2 450-624 oligomerizes and undergoes temperature-responsive liquid-liquid phase transitions following a closed-loop temperature-concentration phase diagram. We recently showed that disease-linked mutations to UBQLN2 450-624 impart highly varying effects to its phase behavior, ranging from little change to significant decrease of saturation concentration and formation of gels and aggregates. However, how single mutations lead to these properties is unknown. Here, we use UBQLN2 450-624 as a model system to study the sequence determinants of phase separation. We hypothesized that UBQLN2 450-624 regions previously identified to promote its oligomerization are the “stickers” that drive interchain interactions and phase separation. We systematically investigated how phase behavior is affected by all 19 possible single amino acid substitutions at three sticker and two “spacer” (sequences separating stickers) positions. Overall, substitutions to stickers, but not spacers, substantially altered the shape of the phase diagram. Within the sticker regions, increasing hydrophobicity decreased saturation concentrations at low temperatures and enhanced oligomerization propensity and viscoelasticity of the dense phase. Conversely, substitutions to acidic residues at all positions greatly increased saturation concentrations. Our data demonstrate that single amino acid substitutions follow a molecular code to tune phase transition behavior of biopolymers.
2018
Riley, Julia F; Dao, Thuy P; Castañeda, Carlos A
Cancer Mutations in SPOP Put a Stop to Its Inter-compartmental Hops Journal Article
In: Mol Cell, vol. 72, no. 1, pp. 1–3, 2018, ISSN: 1097-4164.
@article{pmid30290146,
title = {Cancer Mutations in SPOP Put a Stop to Its Inter-compartmental Hops},
author = {Julia F Riley and Thuy P Dao and Carlos A Castañeda},
doi = {10.1016/j.molcel.2018.09.025},
issn = {1097-4164},
year = {2018},
date = {2018-10-01},
journal = {Mol Cell},
volume = {72},
number = {1},
pages = {1--3},
abstract = {In this issue of Molecular Cell, Bouchard et al. (2018) identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this issue of Molecular Cell, Bouchard et al. (2018) identify liquid-liquid phase separation as a mechanism for substrate-triggered localization of SPOP and ubiquitination machinery to different nuclear bodies and describe how cancer mutations disrupt this process.
Dao, Thuy P; Kolaitis, Regina-Maria; Kim, Hong Joo; O’Donovan, Kevin; Martyniak, Brian; Colicino, Erica; Hehnly, Heidi; Taylor, J Paul; Castañeda, Carlos A
Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions Journal Article
In: Mol Cell, vol. 69, no. 6, pp. 965–978.e6, 2018, ISSN: 1097-4164.
@article{pmid29526694,
title = {Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions},
author = {Thuy P Dao and Regina-Maria Kolaitis and Hong Joo Kim and Kevin O'Donovan and Brian Martyniak and Erica Colicino and Heidi Hehnly and J Paul Taylor and Carlos A Castañeda},
doi = {10.1016/j.molcel.2018.02.004},
issn = {1097-4164},
year = {2018},
date = {2018-03-01},
journal = {Mol Cell},
volume = {69},
number = {6},
pages = {965--978.e6},
abstract = {Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2’s ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2’s biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.
2016
Castañeda, Carlos A; Dixon, Emma K; Walker, Olivier; Chaturvedi, Apurva; Nakasone, Mark A; Curtis, Joseph E; Reed, Megan R; Krueger, Susan; Cropp, T Ashton; Fushman, David
Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins Journal Article
In: Structure, vol. 24, no. 3, pp. 423–436, 2016, ISSN: 1878-4186.
@article{pmid26876099,
title = {Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins},
author = {Carlos A Castañeda and Emma K Dixon and Olivier Walker and Apurva Chaturvedi and Mark A Nakasone and Joseph E Curtis and Megan R Reed and Susan Krueger and T Ashton Cropp and David Fushman},
doi = {10.1016/j.str.2016.01.007},
issn = {1878-4186},
year = {2016},
date = {2016-03-01},
journal = {Structure},
volume = {24},
number = {3},
pages = {423--436},
abstract = {Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.
Castañeda, Carlos A; Chaturvedi, Apurva; Camara, Christina M; Curtis, Joseph E; Krueger, Susan; Fushman, David
Linkage-specific conformational ensembles of non-canonical polyubiquitin chains Journal Article
In: Phys Chem Chem Phys, vol. 18, no. 8, pp. 5771–5788, 2016, ISSN: 1463-9084.
@article{pmid26422168,
title = {Linkage-specific conformational ensembles of non-canonical polyubiquitin chains},
author = {Carlos A Castañeda and Apurva Chaturvedi and Christina M Camara and Joseph E Curtis and Susan Krueger and David Fushman},
doi = {10.1039/c5cp04601g},
issn = {1463-9084},
year = {2016},
date = {2016-02-01},
journal = {Phys Chem Chem Phys},
volume = {18},
number = {8},
pages = {5771--5788},
abstract = {Polyubiquitination is a critical protein post-translational modification involved in a variety of processes in eukaryotic cells. The molecular basis for selective recognition of the polyubiquitin signals by cellular receptors is determined by the conformations polyubiquitin chains adopt; this has been demonstrated for K48- and K63-linked chains. Recent studies of the so-called non-canonical chains (linked via K6, K11, K27, K29, or K33) suggest they play important regulatory roles in growth, development, and immune system pathways, but biophysical studies are needed to elucidate the physical/structural basis of their interactions with receptors. A first step towards this goal is characterization of the conformations these chains adopt in solution. We assembled diubiquitins (Ub2) comprised of every lysine linkage. Using solution NMR measurements, small-angle neutron scattering (SANS), and in silico ensemble generation, we determined population-weighted conformational ensembles that shed light on the structure and dynamics of the non-canonical polyubiquitin chains. We found that polyubiquitin is conformationally heterogeneous, and each chain type exhibits unique conformational ensembles. For example, K6-Ub2 and K11-Ub2 (at physiological salt concentration) are in dynamic equilibrium between at least two conformers, where one exhibits a unique Ub/Ub interface, distinct from that observed in K48-Ub2 but similar to crystal structures of these chains. Conformers for K29-Ub2 and K33-Ub2 resemble recent crystal structures in the ligand-bound state. Remarkably, a number of diubiquitins adopt conformers similar to K48-Ub2 or K63-Ub2, suggesting potential overlap of biological function among different lysine linkages. These studies highlight the potential power of determining function from elucidation of conformational states.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Polyubiquitination is a critical protein post-translational modification involved in a variety of processes in eukaryotic cells. The molecular basis for selective recognition of the polyubiquitin signals by cellular receptors is determined by the conformations polyubiquitin chains adopt; this has been demonstrated for K48- and K63-linked chains. Recent studies of the so-called non-canonical chains (linked via K6, K11, K27, K29, or K33) suggest they play important regulatory roles in growth, development, and immune system pathways, but biophysical studies are needed to elucidate the physical/structural basis of their interactions with receptors. A first step towards this goal is characterization of the conformations these chains adopt in solution. We assembled diubiquitins (Ub2) comprised of every lysine linkage. Using solution NMR measurements, small-angle neutron scattering (SANS), and in silico ensemble generation, we determined population-weighted conformational ensembles that shed light on the structure and dynamics of the non-canonical polyubiquitin chains. We found that polyubiquitin is conformationally heterogeneous, and each chain type exhibits unique conformational ensembles. For example, K6-Ub2 and K11-Ub2 (at physiological salt concentration) are in dynamic equilibrium between at least two conformers, where one exhibits a unique Ub/Ub interface, distinct from that observed in K48-Ub2 but similar to crystal structures of these chains. Conformers for K29-Ub2 and K33-Ub2 resemble recent crystal structures in the ligand-bound state. Remarkably, a number of diubiquitins adopt conformers similar to K48-Ub2 or K63-Ub2, suggesting potential overlap of biological function among different lysine linkages. These studies highlight the potential power of determining function from elucidation of conformational states.
2015
Ha, Jeung-Hoi; Karchin, Joshua M; Walker-Kopp, Nancy; Castañeda, Carlos A; Loh, Stewart N
Engineered Domain Swapping as an On/Off Switch for Protein Function Journal Article
In: Chem Biol, vol. 22, no. 10, pp. 1384–1393, 2015, ISSN: 1879-1301.
@article{pmid26496687,
title = {Engineered Domain Swapping as an On/Off Switch for Protein Function},
author = {Jeung-Hoi Ha and Joshua M Karchin and Nancy Walker-Kopp and Carlos A Castañeda and Stewart N Loh},
doi = {10.1016/j.chembiol.2015.09.007},
issn = {1879-1301},
year = {2015},
date = {2015-10-01},
journal = {Chem Biol},
volume = {22},
number = {10},
pages = {1384--1393},
abstract = {Domain swapping occurs when identical proteins exchange segments in reciprocal fashion. Natural swapping mechanisms remain poorly understood, and engineered swapping has the potential for creating self-assembling biomaterials that encode for emergent functions. We demonstrate that induced swapping can be used to regulate the function of a target protein. Swapping is triggered by inserting a "lever" protein (ubiquitin) into one of four loops of the ribose binding protein (RBP) target. The lever splits the target, forcing RBP to refold in trans to generate swapped oligomers. Identical RBP-ubiquitin fusions form homo-swapped complexes with the ubiquitin domain acting as the hinge. Surprisingly, some pairs of non-identical fusions swap more efficiently with each other than they do with themselves. Nuclear magnetic resonance experiments reveal that the hinge of these hetero-swapped complexes maps to a region of RBP distant from both ubiquitins. This design is expected to be applicable to other proteins to convert them into functional switches.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Domain swapping occurs when identical proteins exchange segments in reciprocal fashion. Natural swapping mechanisms remain poorly understood, and engineered swapping has the potential for creating self-assembling biomaterials that encode for emergent functions. We demonstrate that induced swapping can be used to regulate the function of a target protein. Swapping is triggered by inserting a “lever” protein (ubiquitin) into one of four loops of the ribose binding protein (RBP) target. The lever splits the target, forcing RBP to refold in trans to generate swapped oligomers. Identical RBP-ubiquitin fusions form homo-swapped complexes with the ubiquitin domain acting as the hinge. Surprisingly, some pairs of non-identical fusions swap more efficiently with each other than they do with themselves. Nuclear magnetic resonance experiments reveal that the hinge of these hetero-swapped complexes maps to a region of RBP distant from both ubiquitins. This design is expected to be applicable to other proteins to convert them into functional switches.