2026
Haws, William; Dao, Thuy P.; Varner, Bridget; Jones, Holly B.; Brown, Mallory P.; Castañeda, Carlos A.
STI1 domains coordinate partitioning of UBQLN2 into stress-induced condensates Unpublished
bioRxiv, 2026.
@unpublished{Haws2026,
title = {STI1 domains coordinate partitioning of UBQLN2 into stress-induced condensates},
author = {William Haws and Thuy P. Dao and Bridget Varner and Holly B. Jones and Mallory P. Brown and Carlos A. Castañeda},
url = {http://biorxiv.org/lookup/doi/10.64898/2026.04.01.715099},
doi = {10.64898/2026.04.01.715099},
year = {2026},
date = {2026-04-03},
publisher = {openRxiv},
abstract = {Abstract
UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation
in vitro
and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that
in vitro
phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions.
Highlights
UBQLN2 is recruited to both stress granules and puncta formed by the autophagy receptor protein p62 in response to heat stress.
Both endogenous and overexpressed UBQLN2 tend to colocalize with p62 puncta in cells where p62 is abundant, even without acute stress treatment.
Using an extensive domain deletion library, ability of UBQLN2 constructs to form puncta correlates with their
in vitro
phase separation propensity in the absence of stress.
Each of the two STI1 domains contribute non-redundantly to formation of UBQLN2 puncta in response to heat stress.
The STI1-II domain is independently required for UBQLN2 oligomerization, phase separation, and puncta formation.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation
and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that
phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions.
Using an extensive domain deletion library, ability of UBQLN2 constructs to form puncta correlates with their
phase separation propensity in the absence of stress.
Acharya, Nirbhik; Daniel, Emily A; Dao, Thuy P; Niblo, Jessica K; Mulvey, Erin O; Sukenik, Shahar; Kraut, Daniel A; Roelofs, Jeroen; Castañeda, Carlos A
STI1 domain engages transient helices to mediate Dsk2 phase separation and proteasome condensation Journal Article
In: EMBO J, vol. 45, no. 8, pp. 2712–2738, 2026, ISSN: 1460-2075.
@article{pmid41673446,
title = {STI1 domain engages transient helices to mediate Dsk2 phase separation and proteasome condensation},
author = {Nirbhik Acharya and Emily A Daniel and Thuy P Dao and Jessica K Niblo and Erin O Mulvey and Shahar Sukenik and Daniel A Kraut and Jeroen Roelofs and Carlos A Castañeda},
doi = {10.1038/s44318-026-00696-1},
issn = {1460-2075},
year = {2026},
date = {2026-04-01},
journal = {EMBO J},
volume = {45},
number = {8},
pages = {2712--2738},
abstract = {Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and polyubiquitinated substrates under stress conditions. Here, we identify the chaperone-binding STI1 domain as the main driver of Dsk2 self-association and phase separation. Using nuclear magnetic resonance (NMR) spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces Dsk2's propensity to form condensates. In vivo, perturbing STI1-helix interactions, specifically removal of the transient helices, reduces the formation of azide stress-induced Dsk2/proteasome condensates, in line with our in vitro results. Modeling of Dsk2 STI1-helix interactions reveals a binding mode reminiscent of chaperone STI1/DP2 domains interacting with client helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and polyubiquitinated substrates under stress conditions. Here, we identify the chaperone-binding STI1 domain as the main driver of Dsk2 self-association and phase separation. Using nuclear magnetic resonance (NMR) spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces Dsk2’s propensity to form condensates. In vivo, perturbing STI1-helix interactions, specifically removal of the transient helices, reduces the formation of azide stress-induced Dsk2/proteasome condensates, in line with our in vitro results. Modeling of Dsk2 STI1-helix interactions reveals a binding mode reminiscent of chaperone STI1/DP2 domains interacting with client helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.
Niblo, Jessica K.; Acharya, Nirbhik; Watkins, Maxwell B.; Castañeda, Carlos A.; Sukenik, Shahar
Intramolecular interactions between folded and disordered regions shape ubiquilin structure and function Unpublished
bioRxiv, 2026.
@unpublished{Niblo2026,
title = {Intramolecular interactions between folded and disordered regions shape ubiquilin structure and function},
author = {Jessica K. Niblo and Nirbhik Acharya and Maxwell B. Watkins and Carlos A. Castañeda and Shahar Sukenik},
url = {http://biorxiv.org/lookup/doi/10.64898/2026.03.13.711692},
doi = {10.64898/2026.03.13.711692},
year = {2026},
date = {2026-03-17},
publisher = {openRxiv},
abstract = {Abstract
Multidomain proteins consist of folded domains connected by intrinsically disordered regions. The flexibility afforded by the disordered regions coupled to the structure and surface chemistry of folded regions allows for unique structural and functional features in these proteins. Yet how intramolecular interactions between disordered regions and folded domains affect multidomain protein structure and function remain poorly understood. Here we use a range of biophysical and computational approaches to measure the intramolecular interactions between the folded domains and disordered regions of ubiquilins (UBQLNs) - essential components of protein quality control that shuttle poly-ubiquitinated client proteins to proteasomal degradation or autophagy. Starting with the yeast UBQLN homolog Dsk2, we find that interactions between two folded domains located at the opposite ends of UBQLN bring about a closed conformation. The prevalence of this closed conformation, however, is modulated by intramolecular interactions involving the disordered regions and folded STI1 domain at the center of the protein. Simulations and analysis of UBQLN homologs across multiple eukaryotic lineages reveals that these disordered:folded domain interactions exist in some UBQLN homologs but are absent in others, indicating possible fundamental differences in function among proteins with the same multidomain architecture. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Whited, A. M.; DeLear, Patrick; Thomas, Ezekiel C.; Allen, Jeffre; Ferrer-Imbert, Génesis; Acharya, Nirbhik; Castañeda, Carlos A.; Sept, David; Moore, Jeffrey K.; Hough, Loren E.
Tubulin C-terminal tails are pH sensors that regulate microtubule function Unpublished
bioRxiv, 2026.
@unpublished{Whited2026,
title = {Tubulin C-terminal tails are pH sensors that regulate microtubule function},
author = {A.M. Whited and Patrick DeLear and Ezekiel C. Thomas and Jeffre Allen and Génesis Ferrer-Imbert and Nirbhik Acharya and Carlos A. Castañeda and David Sept and Jeffrey K. Moore and Loren E. Hough},
url = {http://biorxiv.org/lookup/doi/10.64898/2026.03.06.710195},
doi = {10.64898/2026.03.06.710195},
year = {2026},
date = {2026-03-08},
publisher = {openRxiv},
abstract = {Abstract
Changes in intracellular pH are critical for maintaining homeostasis, mediating signaling pathways, and enabling cellular responses to stress, injury, and disease. There is increasing evidence that clusters of acidic residues, primarily glutamates, are both highly prevalent and conserved in disordered regions of proteins and can play an important role in cellular pH response. Tubulin C-terminal tails (CTTs) are glutamate rich regions which protrude from the microtubule surface. These tails are a primary site of for both post-translational modifications and binding of microtubule-associated proteins. Motivated by these observations, we measured the pH response of tubulin CTTs using NMR spectroscopy, circular dichroism, and computational simulations. We find that glutamate residues in CTTs taken from organisms across eukaryotes exhibit a robust upshift in their p
K
a
values, that the sequential context of glutamate residues creates hot spots for protonation, and that hydrogen bonding between side chains stabilizes interactions that alter the conformation of the CTT. To determine whether the CTT pH response plays a potentially important role in microtubule interactions, we measured the pH dependence of the binding of the yeast kinesin-5, Cin8, to microtubules. We find that Cin8 binding is modulated by pH in a CTT-dependent manner. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish that pH can regulate interactions at the microtubule surface.
Significance Statement
Variation in cellular pH is important for cell function in changing environmental conditions or developmental states. Here we probe protonation of the glutamate-rich C-terminal tails of tubulin, revealing the existence of and mechanism driving the anomalously high pH response and subsequent regulation of microtubule binding. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish pH-based regulation of interactions at the microtubule surface.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Changes in intracellular pH are critical for maintaining homeostasis, mediating signaling pathways, and enabling cellular responses to stress, injury, and disease. There is increasing evidence that clusters of acidic residues, primarily glutamates, are both highly prevalent and conserved in disordered regions of proteins and can play an important role in cellular pH response. Tubulin C-terminal tails (CTTs) are glutamate rich regions which protrude from the microtubule surface. These tails are a primary site of for both post-translational modifications and binding of microtubule-associated proteins. Motivated by these observations, we measured the pH response of tubulin CTTs using NMR spectroscopy, circular dichroism, and computational simulations. We find that glutamate residues in CTTs taken from organisms across eukaryotes exhibit a robust upshift in their p
values, that the sequential context of glutamate residues creates hot spots for protonation, and that hydrogen bonding between side chains stabilizes interactions that alter the conformation of the CTT. To determine whether the CTT pH response plays a potentially important role in microtubule interactions, we measured the pH dependence of the binding of the yeast kinesin-5, Cin8, to microtubules. We find that Cin8 binding is modulated by pH in a CTT-dependent manner. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish that pH can regulate interactions at the microtubule surface.
Kurbah, Iladeiti; Castañeda, Carlos A; Fushman, David
Amide H and N NMR signal assignments of all naturally-occurring di-ubiquitins Journal Article
In: Biomol NMR Assign, vol. 20, no. 1, 2026, ISSN: 1874-270X.
@article{pmid41741820,
title = {Amide H and N NMR signal assignments of all naturally-occurring di-ubiquitins},
author = {Iladeiti Kurbah and Carlos A Castañeda and David Fushman},
doi = {10.1007/s12104-026-10261-w},
issn = {1874-270X},
year = {2026},
date = {2026-02-01},
journal = {Biomol NMR Assign},
volume = {20},
number = {1},
abstract = {Ubiquitin acts as a building block for a wide variety of poly-ubiquitin chains. Decoding the role of poly-ubiquitin chains in different cellular processes remains an active area of research. Here, we report amide H and N signal assignments of each ubiquitin unit in di-ubiquitins of all seven lysine linkages and in M1-linked di-ubiquitin determined by our lab over the last decade. These assignments can aid in NMR studies of the structure, dynamics, and function of various di-ubiquitins. Comparison of the NMR resonance assignments among all the di-ubiquitins revealed linkage-specific chemical shifts and isopeptide signals that can be used as "fingerprints" to directly identify using NMR spectroscopy the linkage type in a di-ubiquitin and potentially longer poly-ubiquitin chains. Our data highlight both the similarities and dissimilarities of NMR signals of ubiquitin units in di-ubiquitins of different linkages, as well as the importance of selective isotopic labeling of specific ubiquitin units in a poly-ubiquitin chain for NMR studies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ubiquitin acts as a building block for a wide variety of poly-ubiquitin chains. Decoding the role of poly-ubiquitin chains in different cellular processes remains an active area of research. Here, we report amide H and N signal assignments of each ubiquitin unit in di-ubiquitins of all seven lysine linkages and in M1-linked di-ubiquitin determined by our lab over the last decade. These assignments can aid in NMR studies of the structure, dynamics, and function of various di-ubiquitins. Comparison of the NMR resonance assignments among all the di-ubiquitins revealed linkage-specific chemical shifts and isopeptide signals that can be used as “fingerprints” to directly identify using NMR spectroscopy the linkage type in a di-ubiquitin and potentially longer poly-ubiquitin chains. Our data highlight both the similarities and dissimilarities of NMR signals of ubiquitin units in di-ubiquitins of different linkages, as well as the importance of selective isotopic labeling of specific ubiquitin units in a poly-ubiquitin chain for NMR studies.
2025
Flores, Eduardo; Acharya, Nirbhik; Castañeda, Carlos A; Sukenik, Shahar
Single-point mutations in disordered proteins: Linking sequence, ensemble, and function Journal Article
In: Curr Opin Struct Biol, vol. 91, pp. 102987, 2025, ISSN: 1879-033X.
@article{pmid39914051,
title = {Single-point mutations in disordered proteins: Linking sequence, ensemble, and function},
author = {Eduardo Flores and Nirbhik Acharya and Carlos A Castañeda and Shahar Sukenik},
doi = {10.1016/j.sbi.2025.102987},
issn = {1879-033X},
year = {2025},
date = {2025-04-01},
journal = {Curr Opin Struct Biol},
volume = {91},
pages = {102987},
abstract = {Mutations in genomic DNA often result in single-point missense mutations in proteins. For folded proteins, the functional effect of these missense mutations can often be understood by their impact on structure. However, missense mutations in intrinsically disordered protein regions (IDRs) remain poorly understood. In IDRs, function can depend on the structural ensemble- the collection of accessible, interchanging conformations that is encoded in their amino acid sequence. We argue that, analogously to folded proteins, single-point mutations in IDRs can alter their structural ensemble, and consequently alter their biological function. To make this argument, we first provide experimental evidence from the literature showcasing how single-point missense mutations in IDRs affect their ensemble dimensions. Then, we use genomic data from patients to show that disease-linked missense mutations occurring in IDRs can, in many cases, significantly alter IDR structural ensembles. We hope this analysis prompts further study of disease-linked, single-point mutations in IDRs.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mutations in genomic DNA often result in single-point missense mutations in proteins. For folded proteins, the functional effect of these missense mutations can often be understood by their impact on structure. However, missense mutations in intrinsically disordered protein regions (IDRs) remain poorly understood. In IDRs, function can depend on the structural ensemble- the collection of accessible, interchanging conformations that is encoded in their amino acid sequence. We argue that, analogously to folded proteins, single-point mutations in IDRs can alter their structural ensemble, and consequently alter their biological function. To make this argument, we first provide experimental evidence from the literature showcasing how single-point missense mutations in IDRs affect their ensemble dimensions. Then, we use genomic data from patients to show that disease-linked missense mutations occurring in IDRs can, in many cases, significantly alter IDR structural ensembles. We hope this analysis prompts further study of disease-linked, single-point mutations in IDRs.
Acharya, Nirbhik; Daniel, Emily A.; Dao, Thuy P.; Niblo, Jessica K.; Mulvey, Erin; Sukenik, Shahar; Kraut, Daniel A.; Roelofs, Jeroen; Castañeda, Carlos A.
bioRxiv, 2025.
@unpublished{Acharya2025,
title = {STI1 domain dynamically engages transient helices in disordered regions to drive self-association and phase separation of yeast ubiquilin Dsk2},
author = {Nirbhik Acharya and Emily A. Daniel and Thuy P. Dao and Jessica K. Niblo and Erin Mulvey and Shahar Sukenik and Daniel A. Kraut and Jeroen Roelofs and Carlos A. Castañeda},
url = {http://biorxiv.org/lookup/doi/10.1101/2025.03.14.643327},
doi = {10.1101/2025.03.14.643327},
year = {2025},
date = {2025-03-14},
publisher = {openRxiv},
abstract = {Abstract
Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation
in vitro
. Using NMR spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically-disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces the propensity for Dsk2 to phase separate.
In vivo
, removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.
Highlights
The intrinsically disordered regions of Dsk2 harbor transient helices that regulate protein properties via interactions with the STI1 domain.
The STI1 domain is a significant driver of Dsk2 self-association and phase separation
in vitro
.
Dsk2 colocalizes with ubiquitinated substrates and proteasome in reconstituted condensates.
Absence of Dsk2 STI1 domain in stressed yeast cells promotes formation of proteasome condensates coupled with upregulation of polyubiquitinated substrates.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation
. Using NMR spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically-disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces the propensity for Dsk2 to phase separate.
, removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.
The STI1 domain is a significant driver of Dsk2 self-association and phase separation
.
Rajendran, Anitha; Castañeda, Carlos A
Protein quality control machinery: regulators of condensate architecture and functionality Journal Article
In: Trends Biochem Sci, vol. 50, no. 2, pp. 106–120, 2025, ISSN: 0968-0004.
@article{pmid39755440,
title = {Protein quality control machinery: regulators of condensate architecture and functionality},
author = {Anitha Rajendran and Carlos A Castañeda},
doi = {10.1016/j.tibs.2024.12.003},
issn = {0968-0004},
year = {2025},
date = {2025-02-01},
journal = {Trends Biochem Sci},
volume = {50},
number = {2},
pages = {106--120},
abstract = {Protein quality control (PQC) mechanisms including the ubiquitin (Ub)-proteasome system (UPS), autophagy, and chaperone-mediated refolding are essential to maintain protein homeostasis in cells. Recent studies show that these PQC mechanisms are further modulated by biomolecular condensates that sequester PQC components and compartmentalize reactions. Accumulating evidence points towards the PQC machinery playing a pivotal role in regulating the assembly, disassembly, and viscoelastic properties of several condensates. Here, we discuss how the PQC machinery can form their own condensates and also be recruited to known condensates under physiological or stress-induced conditions. We present molecular insights into how the multivalent architecture of polyUb chains, Ub-binding adaptor proteins, and other PQC machinery contribute to condensate assembly, leading to the regulation of downstream PQC outcomes and therapeutic potential.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Protein quality control (PQC) mechanisms including the ubiquitin (Ub)-proteasome system (UPS), autophagy, and chaperone-mediated refolding are essential to maintain protein homeostasis in cells. Recent studies show that these PQC mechanisms are further modulated by biomolecular condensates that sequester PQC components and compartmentalize reactions. Accumulating evidence points towards the PQC machinery playing a pivotal role in regulating the assembly, disassembly, and viscoelastic properties of several condensates. Here, we discuss how the PQC machinery can form their own condensates and also be recruited to known condensates under physiological or stress-induced conditions. We present molecular insights into how the multivalent architecture of polyUb chains, Ub-binding adaptor proteins, and other PQC machinery contribute to condensate assembly, leading to the regulation of downstream PQC outcomes and therapeutic potential.
2024
Valentino, Isabella M.; Llivicota-Guaman, Jeniffer G.; Dao, Thuy P.; Mulvey, Erin O.; Lehman, Andrew M.; Galagedera, Sarasi K. K.; Mallon, Erica L.; Castañeda, Carlos A.; Kraut, Daniel A.
Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate Journal Article
In: Proc. Natl. Acad. Sci. U.S.A., vol. 121, no. 33, 2024, ISSN: 1091-6490.
@article{Valentino2024b,
title = {Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate},
author = {Isabella M. Valentino and Jeniffer G. Llivicota-Guaman and Thuy P. Dao and Erin O. Mulvey and Andrew M. Lehman and Sarasi K. K. Galagedera and Erica L. Mallon and Carlos A. Castañeda and Daniel A. Kraut},
doi = {10.1073/pnas.2405964121},
issn = {1091-6490},
year = {2024},
date = {2024-08-13},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {121},
number = {33},
publisher = {Proceedings of the National Academy of Sciences},
abstract = {Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2’s phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code. },
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Alisafaei, Farid; Mandal, Kalpana; Saldanha, Renita; Swoger, Maxx; Yang, Haiqian; Shi, Xuechen; Guo, Ming; Hehnly, Heidi; Castañeda, Carlos A; Janmey, Paul A; Patteson, Alison E; Shenoy, Vivek B
Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks Journal Article
In: Commun Biol, vol. 7, no. 1, pp. 658, 2024, ISSN: 2399-3642.
@article{pmid38811770,
title = {Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks},
author = {Farid Alisafaei and Kalpana Mandal and Renita Saldanha and Maxx Swoger and Haiqian Yang and Xuechen Shi and Ming Guo and Heidi Hehnly and Carlos A Castañeda and Paul A Janmey and Alison E Patteson and Vivek B Shenoy},
doi = {10.1038/s42003-024-06366-4},
issn = {2399-3642},
year = {2024},
date = {2024-05-01},
journal = {Commun Biol},
volume = {7},
number = {1},
pages = {658},
abstract = {The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin’s effects on the transmission of cell contractile forces to the extracellular matrix.
Safren, Nathaniel; Dao, Thuy P; Mohan, Harihar Milaganur; Huang, Camellia; Trotter, Bryce; Castañeda, Carlos A; Paulson, Henry; Barmada, Sami; Sharkey, Lisa M
Pathogenic mutations in UBQLN2 exhibit diverse aggregation propensity and neurotoxicity Journal Article
In: Sci Rep, vol. 14, no. 1, pp. 6049, 2024, ISSN: 2045-2322.
@article{pmid38472280,
title = {Pathogenic mutations in UBQLN2 exhibit diverse aggregation propensity and neurotoxicity},
author = {Nathaniel Safren and Thuy P Dao and Harihar Milaganur Mohan and Camellia Huang and Bryce Trotter and Carlos A Castañeda and Henry Paulson and Sami Barmada and Lisa M Sharkey},
doi = {10.1038/s41598-024-55582-9},
issn = {2045-2322},
year = {2024},
date = {2024-03-01},
urldate = {2024-03-01},
journal = {Sci Rep},
volume = {14},
number = {1},
pages = {6049},
abstract = {The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.
2023
Dao, Thuy P; Rajendran, Anitha; Galagedera, Sarasi K K; Haws, William; Castañeda, Carlos A
Short disordered N-termini & proline-rich domain are major regulators of UBQLN1/2/4 phase separation Journal Article
In: Biophys J, 2023, ISSN: 1542-0086.
@article{pmid38041404,
title = {Short disordered N-termini & proline-rich domain are major regulators of UBQLN1/2/4 phase separation},
author = {Thuy P Dao and Anitha Rajendran and Sarasi K K Galagedera and William Haws and Carlos A Castañeda},
doi = {10.1016/j.bpj.2023.11.3401},
issn = {1542-0086},
year = {2023},
date = {2023-11-01},
journal = {Biophys J},
abstract = {Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts.
Galagedera, Sarasi K K; Dao, Thuy P; Enos, Suzanne E; Chaudhuri, Antara; Schmit, Jeremy D; Castañeda, Carlos A
Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units Journal Article
In: Proc Natl Acad Sci U S A, vol. 120, no. 42, pp. e2306638120, 2023, ISSN: 1091-6490.
@article{pmid37824531,
title = {Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units},
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 = {10.1073/pnas.2306638120},
issn = {1091-6490},
year = {2023},
date = {2023-10-01},
journal = {Proc Natl Acad Sci U S A},
volume = {120},
number = {42},
pages = {e2306638120},
abstract = {Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains 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. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by 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. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains 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. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by 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. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
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.
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.
Caselle, Elizabeth A; Yoon, Jennifer H; Bhattacharya, Sagar; Rempillo, Joel J L; Lengyel, Zsófia; D’Souza, Areetha; Moroz, Yurii S; Tolbert, Patricia L; Volkov, Alexander N; Forconi, Marcello; Castañeda, Carlos A; Makhlynets, Olga V; Korendovych, Ivan V
Kemp Eliminases of the AlleyCat Family Possess High Substrate Promiscuity Journal Article
In: ChemCatChem, vol. 11, no. 5, pp. 1425–1430, 2019, ISSN: 1867-3880.
@article{pmid31788134,
title = {Kemp Eliminases of the AlleyCat Family Possess High Substrate Promiscuity},
author = {Elizabeth A Caselle and Jennifer H Yoon and Sagar Bhattacharya and Joel J L Rempillo and Zsófia Lengyel and Areetha D'Souza and Yurii S Moroz and Patricia L Tolbert and Alexander N Volkov and Marcello Forconi and Carlos A Castañeda and Olga V Makhlynets and Ivan V Korendovych},
doi = {10.1002/cctc.201801994},
issn = {1867-3880},
year = {2019},
date = {2019-03-01},
journal = {ChemCatChem},
volume = {11},
number = {5},
pages = {1425--1430},
abstract = {Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pK.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pK.
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.
2017
Hiebler, Katharina; Lengyel, Zsófia; Castañeda, Carlos A; Makhlynets, Olga V
Functional tuning of the catalytic residue pK in a de novo designed esterase Journal Article
In: Proteins, vol. 85, no. 9, pp. 1656–1665, 2017, ISSN: 1097-0134.
@article{pmid28544090,
title = {Functional tuning of the catalytic residue pK in a de novo designed esterase},
author = {Katharina Hiebler and Zsófia Lengyel and Carlos A Castañeda and Olga V Makhlynets},
doi = {10.1002/prot.25321},
issn = {1097-0134},
year = {2017},
date = {2017-09-01},
journal = {Proteins},
volume = {85},
number = {9},
pages = {1656--1665},
abstract = {AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK value of this residue in water. We determined that a short-range favorable interaction with Glu contributes to the elevated pK of His . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK of its active nucleophile, His , by 0.7 units. As a direct result of the decrease in the His pK value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK value of this residue in water. We determined that a short-range favorable interaction with Glu contributes to the elevated pK of His . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK of its active nucleophile, His , by 0.7 units. As a direct result of the decrease in the His pK value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.
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.
2013
Wolking, Stefan; Lerche, Holger; Dihné, Marcel
Episodic itch in a case of spinal glioma Journal Article
In: BMC Neurol, vol. 13, pp. 124, 2013, ISSN: 1471-2377.
@article{pmid24059641,
title = {Episodic itch in a case of spinal glioma},
author = {Stefan Wolking and Holger Lerche and Marcel Dihné},
doi = {10.1186/1471-2377-13-124},
issn = {1471-2377},
year = {2013},
date = {2013-09-01},
journal = {BMC Neurol},
volume = {13},
pages = {124},
abstract = {BACKGROUND: Itch is a frequent complaint reported by patients and is usually ascribed to dermatological or metabolic causes. In neurological disorders, however, it is a very unusual symptom and thus its neurological aetiology is likely to be overlooked. There are only very few reports about permanent itch related to lesions of the central nervous system. To our knowledge we report the first case of episodic itch associated with a central nervous lesion.nnCASE PRESENTATION: A 74-year-old female suffered from long-standing episodes of itch of the dermatomes C2 to C6 on the right side that was refractory to any treatment. On occurrence it propagated in a proximal to distal fashion. Between the episodes the patient was asymptomatic. MRI of the cervical spine uncovered a spinal glioma that matched the location of the symptoms. Treatment with gabapentin led to a prompt reduction of the symptoms.nnCONCLUSION: Patients with intractable pruritus and dermatomal presentation ought to undergo neurological examination and spinal cord imaging. Thus, ongoing frustrating and sometimes even harmful treatment trials could be avoided.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Itch is a frequent complaint reported by patients and is usually ascribed to dermatological or metabolic causes. In neurological disorders, however, it is a very unusual symptom and thus its neurological aetiology is likely to be overlooked. There are only very few reports about permanent itch related to lesions of the central nervous system. To our knowledge we report the first case of episodic itch associated with a central nervous lesion.nnCASE PRESENTATION: A 74-year-old female suffered from long-standing episodes of itch of the dermatomes C2 to C6 on the right side that was refractory to any treatment. On occurrence it propagated in a proximal to distal fashion. Between the episodes the patient was asymptomatic. MRI of the cervical spine uncovered a spinal glioma that matched the location of the symptoms. Treatment with gabapentin led to a prompt reduction of the symptoms.nnCONCLUSION: Patients with intractable pruritus and dermatomal presentation ought to undergo neurological examination and spinal cord imaging. Thus, ongoing frustrating and sometimes even harmful treatment trials could be avoided.
0000
Kurbah, Iladeiti; Castañeda, Carlos A.; Fushman, David
Amide 1H and 15N NMR signal assignments of all naturally-occurring di-ubiquitins Journal Article
In: Biomol NMR Assign, vol. 20, no. 1, 0000, ISSN: 1874-270X.
@article{Kurbah2026,
title = {Amide 1H and 15N NMR signal assignments of all naturally-occurring di-ubiquitins},
author = {Iladeiti Kurbah and Carlos A. Castañeda and David Fushman},
doi = {10.1007/s12104-026-10261-w},
issn = {1874-270X},
journal = {Biomol NMR Assign},
volume = {20},
number = {1},
publisher = {Springer Science and Business Media LLC},
abstract = {Abstract
Ubiquitin acts as a building block for a wide variety of poly-ubiquitin chains. Decoding the role of poly-ubiquitin chains in different cellular processes remains an active area of research. Here, we report amide
1
H and
15
N signal assignments of each ubiquitin unit in di-ubiquitins of all seven lysine linkages and in M1-linked di-ubiquitin determined by our lab over the last decade. These assignments can aid in NMR studies of the structure, dynamics, and function of various di-ubiquitins. Comparison of the NMR resonance assignments among all the di-ubiquitins revealed linkage-specific chemical shifts and isopeptide signals that can be used as “fingerprints” to directly identify using NMR spectroscopy the linkage type in a di-ubiquitin and potentially longer poly-ubiquitin chains. Our data highlight both the similarities and dissimilarities of NMR signals of ubiquitin units in di-ubiquitins of different linkages, as well as the importance of selective isotopic labeling of specific ubiquitin units in a poly-ubiquitin chain for NMR studies.
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Ubiquitin acts as a building block for a wide variety of poly-ubiquitin chains. Decoding the role of poly-ubiquitin chains in different cellular processes remains an active area of research. Here, we report amide
H and
N signal assignments of each ubiquitin unit in di-ubiquitins of all seven lysine linkages and in M1-linked di-ubiquitin determined by our lab over the last decade. These assignments can aid in NMR studies of the structure, dynamics, and function of various di-ubiquitins. Comparison of the NMR resonance assignments among all the di-ubiquitins revealed linkage-specific chemical shifts and isopeptide signals that can be used as “fingerprints” to directly identify using NMR spectroscopy the linkage type in a di-ubiquitin and potentially longer poly-ubiquitin chains. Our data highlight both the similarities and dissimilarities of NMR signals of ubiquitin units in di-ubiquitins of different linkages, as well as the importance of selective isotopic labeling of specific ubiquitin units in a poly-ubiquitin chain for NMR studies.