Publications
23 results found
Morten MJ, Sirvio L, Rupawala H, et al., 2022, Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies., Proceedings of the National Academy of Sciences of USA, Vol: 119, Pages: 1-12, ISSN: 0027-8424
Protein aggregation is a hallmark of major neurodegenerative disorders. Increasing data suggest that smaller aggregates cause higher toxic response than filamentous aggregates (fibrils). However, the size of small aggregates has challenged their detection within biologically relevant environments. Here, we report approaches to quantitatively super-resolve aggregates in live cells and ex vivo brain tissues. We show that Amytracker 630 (AT630), a commercial aggregate-activated fluorophore, has outstanding photophysical properties that enable super-resolution imaging of α-synuclein, tau, and amyloid-β aggregates, achieving ∼4 nm precision. Applying AT630 to AppNL-G-F mouse brain tissues or aggregates extracted from a Parkinson's disease donor, we demonstrate excellent agreement with antibodies specific for amyloid-β or α-synuclein, respectively, confirming the specificity of AT630. Subsequently, we use AT630 to reveal a linear relationship between α-synuclein aggregate size and cellular toxicity and discovered that aggregates smaller than 450 ± 60 nm (aggregate450nm) readily penetrated the plasma membrane. We determine aggregate450nm concentrations in six Parkinson's disease and dementia with Lewy bodies donor samples and show that aggregates in different synucleinopathies demonstrate distinct potency in toxicity. We further show that cell-penetrating aggregates are surrounded by proteasomes, which assemble into foci to gradually process aggregates. Our results suggest that the plasma membrane effectively filters out fibrils but is vulnerable to penetration by aggregates of 450 ± 60 nm. Together, our findings present an exciting strategy to determine specificity of aggregate toxicity within heterogeneous samples. Our approach to quantitatively measure these toxic aggregates in biological environments opens possibilities to molecular examinations of disease mechanisms under physiological conditions.
Meng JX, Zhang Y, Saman D, et al., 2022, Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses, Nature Communications, Vol: 13, Pages: 1-16, ISSN: 2041-1723
Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3β or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.
Mee Hayes E, Sirvio L, Ye Y, 2022, A potential mechanism for targeting aggregates with proteasomes and disaggregases in liquid droplets, Frontiers in Aging Neuroscience, Vol: 14, ISSN: 1663-4365
Insoluble protein deposits are hallmarks of neurodegenerative disorders and common forms of dementia. The aberrant aggregation of misfolded proteins involves a complex cascade of events that occur over time, from the cellular to the clinical phase of neurodegeneration. Declining neuronal health through increased cell stress and loss of protein homeostasis (proteostasis) functions correlate with the accumulation of aggregates. On the cellular level, increasing evidence supports that misfolded proteins may undergo liquid-liquid phase separation (LLPS), which is emerging as an important process to drive protein aggregation. Studying the reverse process of aggregate disassembly and degradation has only recently gained momentum, following reports of enzymes with distinct aggregate-disassembly activities. In this review, we will discuss how the ubiquitin-proteasome system and disaggregation machineries such as VCP/p97 and HSP70 system may disassemble and/or degrade protein aggregates. In addition to their canonically associated functions, these enzymes appear to share a common feature: reversibly assembling into liquid droplets in an LLPS-driven manner. We review the role of LLPS in enhancing the disassembly of aggregates through locally increasing the concentration of these enzymes and their co-proteins together within droplet structures. We propose that such activity may be achieved through the concerted actions of disaggregase machineries, the ubiquitin-proteasome system and their co-proteins, all of which are condensed within transient aggregate-associated droplets (TAADs), ultimately resulting in aggregate clearance. We further speculate that sustained engagement of these enzymatic activities within TAADs will be detrimental to normal cellular functions, where these activities are required. The possibility of facilitating endogenous disaggregation and degradation activities within TAADs potentially represents a novel target for therapeutic intervention to restore pro
Klenerman D, Meng J, Zhang Y, et al., 2021, Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent inflammatory responses
<jats:title>Abstract</jats:title> <jats:p>Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by PKA in conjugation with either GSK3-β or SAPK4 enabled recombinant wild-type (WT) tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites and the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, in comparison with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4 (TLR4)-dependent inflammatory responses. Together, our results demonstrate that tau hyperphosphorylation is potentially damaging to cells, providing a mechanistic model of how hyperphosphorylation of tau aggregates drives neuroinflammation in tauopathies.</jats:p>
Li B, Ponjavic A, Chen W-H, et al., 2021, Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery, ANALYTICAL CHEMISTRY, Vol: 93, Pages: 4092-4099, ISSN: 0003-2700
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- Citations: 7
Zuo Y, Chong BK, Jiang K, et al., 2020, A general in vitro assay to study enzymatic activities of the ubiquitin system, Biochemistry, Vol: 59, Pages: 851-861, ISSN: 0006-2960
The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the in vitro kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.
Ye Y, Klenerman D, Finley D, 2020, N-Terminal Ubiquitination of Amyloidogenic Proteins Triggers Removal of Their Oligomers by the Proteasome Holoenzyme, Journal of Molecular Biology, Vol: 432, Pages: 585-596, ISSN: 0022-2836
Zuo Y, Chong BK, Jiang K, et al., 2019, A general<i>in vitro</i>assay to study enzymatic activities of the ubiquitin system
<jats:title>Abstract</jats:title><jats:p>The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for protein ubiquitination, thereby controlling cellular activities. Due to the numerous enzymes and processes involved, studies on ubiquitination activities have been challenging. We here report a novel FRET-based assay to study the<jats:italic>in vitro</jats:italic>kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model E1, E2 and E3 enzymes, respectively, we demonstrate that ubiquitination results in decreasing FRET efficiency, from which reaction rates can be determined. Further treatment with USP21, a deubiquitinase, leads to increased FRET efficiency, confirming the reversibility of the assay. We subsequently use this assay to show that increasing the concentration of CHIP or UbcH5 but not Uba1 enhances ubiquitination rates, and develop a novel machine learning approach to model ubiquitination. The overall ubiquitination activity is also increased upon incubation with tau, a substrate of CHIP. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus likely compatible with any E1-E2-E3 combinations.</jats:p>
Ye Y, Klenerman D, Finley D, 2019, N-terminal ubiquitination of amyloidogenic proteins triggers removal of their oligomers by the proteasome holoenzyme
<jats:p>Aggregation of amyloidogenic proteins is an abnormal biological process implicated in neurodegenerative disorders. While the aggregation process of amyloid-forming proteins has been studied extensively, the mechanism of aggregate removal is poorly understood. We recently demonstrated that proteasomes could fragment filamentous aggregates into smaller entities, restricting aggregate size[1]. Here, we show <jats:italic>in vitro</jats:italic> that UBE2W can modify the N-terminus of both αS and tau<jats:sup>K18</jats:sup> with a single ubiquitin moiety. We demonstrate that an engineered N-terminal Ub modification changes the aggregation process of both proteins, resulting in the formation of structurally distinct aggregates. Single-molecule approaches further reveal that the proteasome can target soluble oligomers assembled from Ub-modified proteins independent of its peptidase activity, consistent with our recently reported fibril-fragmenting activity. Based on these results, we propose that proteasomes are able to target oligomers assembled from N-terminally ubiquitinated proteins. Our data suggest a possible disassembly mechanism by which N-terminal ubiquitination and the proteasome may together impede aggregate formation.</jats:p>
Cliffe R, Sang JC, Kundel F, et al., 2019, Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme, Cell Reports, Vol: 26, Pages: 2140-2149.e3, ISSN: 2211-1247
Zhang Y, Lippert AH, Lee JE, et al., 2018, Membrane potential regulates the dynamic localisation of mammalian proteasomes
<jats:title>Abstract</jats:title><jats:p>Proteasomes are molecular machineries responsible for regulated protein degradation and general homeostasis. The distribution of this degradation capacity is reflected by the cellular localisation of proteasomal particles. Here we combine super-resolution imaging, single-particle tracking (SPT) and single-cell patch clamp techniques to investigate the localisation and translocation of endogenous mammalian proteasomes tagged with fluorescent proteins. While proteasomes are found dispersed in the cell without distinct localisation, we detect a higher density of proteasomes in the nucleus compared to the ER and the cytosol. SPT of proteasomes revealed two populations with diffusion coefficients averaging ~4 and ~0.8 μm<jats:sup>2</jats:sup>/s. The ratio between these two populations could be altered upon changed cellular conditions. We further report that proteasomal particles translocate to the cell periphery during hyperpolarisation, while depolarisation re-localises proteasomes to the cell interior. Depolymerising microtubules or actin filaments inhibited this potential-dependent translocation. Our results suggest that at resting membrane potential proteasomes undergo diffusion-based motions, while membrane polarisation may induce cytoskeleton-dependent translocation. Fine-tuning these translocation modes can potentially dedicate proteasomes to degradation activities at distinct subcellular sites.</jats:p>
Ponjavic A, Ye Y, Laue E, et al., 2018, Sensitive light-sheet microscopy in multiwell plates using an AFM cantilever, Biomedical Optics Express, Vol: 9, Pages: 5863-5863, ISSN: 2156-7085
Cliffe R, Sang JC, Kundel F, et al., 2018, Filamentous aggregates are fragmented by the proteasome holoenzyme
<jats:title>Abstract</jats:title><jats:p>Filamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Although there have been numerous studies of the aggregation process <jats:italic>in vitro</jats:italic>, far fewer studies of aggregate disassembly and degradation are available. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. TEM further revealed that these small aggregate species had no distinct structure. The intact proteasome holoenzyme is required to effectively target fibrils. Interestingly, while degradation of monomeric tau was not inhibited by ATPγS, fibril fragmentation was predominantly dependent on the ATPase activity of the proteasome. The proteasome holoenzyme was also found to target fibrils assembled from α-synuclein (αS), suggesting that its fibril fragmenting function may be a general mechanism. The fragmented species produced by the proteasome showed significant toxicity to human cell lines compared to intact fibrils. Together, our results indicate that the proteasome holoenzyme possesses a novel fragmentation function that disassembles large fibrils into smaller and more cytotoxic species.</jats:p>
Iljina M, Tosatto L, Choi ML, et al., 2016, Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein, Scientific Reports, Vol: 6, ISSN: 2045-2322
The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule FÓ§rster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.
Ye Y, 2013, Fluorescent-labelled diubiquitin substrate for a deubiquitinase assay, WO/2011/157982A1; US 20130102012A1; EP2580344A1
The application relates to a substrate for measuring the activity of a deubiquitinating enzyme (DUB), comprising a diubiquitin molecule, wherein an ubiquitin monomer is labeled with a fluorescent label, as well as an assay for DUB enzymes using such substrates.
Ye Y, Blaser G, Horrocks MH, et al., 2012, Ubiquitin chain conformation regulates recognition and activity of interacting proteins, Nature, Vol: 492, Pages: 266-270, ISSN: 0028-0836
Mechanisms of protein recognition have been extensively studied for single-domain proteins1, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins2,3. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended ‘open’ and more compact ‘closed’ conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48–diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.
Agromayor M, Soler N, Caballe A, et al., 2012, The UBAP1 Subunit of ESCRT-I Interacts with Ubiquitin via a SOUBA Domain, STRUCTURE, Vol: 20, Pages: 414-428, ISSN: 0969-2126
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- Citations: 77
KOMANDER DAVID, YE YU, 2011, FLUORESCENT -LABELLED DIUBIQUITIN SUBSTRATE FOR A DEUBIQUITINASE ASSAY
There is described a substrate for measuring the activity of a deubiquitinating enzyme (DUB), comprising a diubiquitin molecule, wherein an ubiquitin monomer is labeled with a fluorescent label, as well as an assay for DUB enzymes using such substrates.
Ye Y, Akutsu M, Reyes-Turcu F, et al., 2011, Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21, EMBO REPORTS, Vol: 12, Pages: 350-357, ISSN: 1469-221X
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- Citations: 125
Akutsu M, Ye Y, Virdee S, et al., 2011, Molecular basis for ubiquitin and ISG15 cross-reactivity in viral ovarian tumor domains, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 2228-2233, ISSN: 0027-8424
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- Citations: 106
Virdee S, Ye Y, Nguyen DP, et al., 2010, Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase, NATURE CHEMICAL BIOLOGY, Vol: 6, Pages: 750-757, ISSN: 1552-4450
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- Citations: 241
Ye Y, Scheel H, Hofmann K, et al., 2009, Dissection of USP catalytic domains reveals five common insertion points, MOLECULAR BIOSYSTEMS, Vol: 5, Pages: 1797-1808, ISSN: 1742-206X
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- Citations: 119
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