scholarly journals Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

2021 ◽  
Author(s):  
Tongyin Zheng ◽  
Carlos A. Castañeda
Keyword(s):  
Quality Control ◽  
Phase Separation ◽  
Liquid Phase ◽  
Protein Quality ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Uba Domain ◽  
Phase Separation Behavior ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

AbstractShuttle 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 intrinsically disordered 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 dropletsin vitroand 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 NMR spectroscopy. To our surprise, aside from well-studied canonical UBL:UBA interactions, there also exist moderate and weak interactions between the UBL and STI1-I/STI1-II domains, and between the UBA domain and the linker connecting the two STI1 regions, respectively. 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 STI1 domains on the phase behavior and physiological functions of UBQLN2.Impact of Work StatementZheng and Castañeda show that interplay between the folded domains and intrinsically disordered regions regulates liquid-liquid phase separation behavior of UBQLN2, a protein quality control (PQC) shuttle protein. Despite their similar size, the folded UBL and UBA domains inhibit and promote phase separation, respectively, due to their previously uncharacterized, asymmetric interactions with the middle intrinsically-disordered region. These results strongly suggest that PQC components, including proteasomal receptors, are likely to modulate UBQLN2 phase separation behavior in cells.

scholarly journals Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

2019 ◽  
Author(s):  
Soumik Ray ◽  
Nitu Singh ◽  
Satyaprakash Pandey ◽  
Rakesh Kumar ◽  
Laxmikant Gadhe ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Amyloid Formation ◽  
Liquid To Solid Transition ◽  
Phase Separation Behavior ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

SUMMARYα-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote α-Syn LLPS and its subsequent maturation. We further demonstrate α-Syn liquid droplet formation in cells, under oxidative stress. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

scholarly journals Transcription Regulators and Membraneless Organelles Challenges to Investigate Them

2021 ◽  
Vol 22 (23) ◽  
pp. 12758
Author(s):  
Katarzyna Sołtys ◽  
Andrzej Ożyhar
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Cellular Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Experimental Systems ◽  
Transcription Regulators ◽  
Crucial Information ◽  
Liquid Liquid Phase Separation

Eukaryotic cells are composed of different bio-macromolecules that are divided into compartments called organelles providing optimal microenvironments for many cellular processes. A specific type of organelles is membraneless organelles. They are formed via a process called liquid–liquid phase separation that is driven by weak multivalent interactions between particular bio-macromolecules. In this review, we gather crucial information regarding different classes of transcription regulators with the propensity to undergo liquid–liquid phase separation and stress the role of intrinsically disordered regions in this phenomenon. We also discuss recently developed experimental systems for studying formation and properties of membraneless organelles.

scholarly journals The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

2020 ◽  
Vol 295 (8) ◽  
pp. 2375-2384 ◽  
Author(s):  
Anastasia C. Murthy ◽  
Nicolas L. Fawzi
Keyword(s):  
Phase Separation ◽  
Nmr Spectroscopy ◽  
Liquid Phase ◽  
Structural Biology ◽  
Liquid Phase Separation ◽  
Biological Processes ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Molecular Aspects ◽  
Liquid Liquid Phase Separation

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.

scholarly journals Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice

2021 ◽  
Vol 7 (1) ◽  
pp. eabd3568
Author(s):  
Nils Schneider ◽  
Franz-Georg Wieland ◽  
Deqiang Kong ◽  
Alexandra A. M. Fischer ◽  
Maximilian Hörner ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Phase Separation ◽  
Liquid Phase ◽  
Mammalian Cells ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gene Switches ◽  
Liquid Liquid Phase Separation

Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

scholarly journals A generic approach for studying the kinetics of liquid-liquid phase separation under near-native conditions

2019 ◽  
Author(s):  
Joris van Lindt ◽  
Anna Bratek-Skicki ◽  
Donya Pakravan ◽  
Ludo Van Den Bosch ◽  
Dominique Maes ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Cell Biology ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Ph Values ◽  
Phase Separation Behavior ◽  
Kinetics Of ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

Understanding the kinetics and underlying physicochemical forces of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for the analysis of often severely aggregation-prone proteins. Frequently applied approaches, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, however, often lead to very different kinetic behaviors. Here we suggest that at extreme pH values even proteins such as the low-complexity domain (LCD) of hnRNPA2, TDP-43, and NUP-98 can be kept in solution, and then their LLPS can be induced by a jump to native pH, resulting in a system that can be easily controlled. This approach represents a generic method for studying LLPS under near native conditions, providing a platform for studying the phase-separation behavior of diverse proteins.

scholarly journals Organella bezbłonowe a separacja faz ciecz-ciecz – metody ich badań

2020 ◽  
Vol 66 (2) ◽  
Author(s):  
Aneta Tarczewska ◽  
Krzysztof Wycisk ◽  
Nikola Sozańska ◽  
Andrzej Ożyhar
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Liquid Liquid Phase Separation ◽  
Disordered Regions

Organella bezbłonowe (MLOs, ang. membraneless organelles) stanowią sporą grupę kompartmentów wewnątrzkomórkowych formowanych na różnych etapach życia komórki. Są one niezbędnymi strukturami umożliwiającymi komórce pełnienie istotnych funkcji życiowych i właściwą odpowiedź na stres. MLOs obecne są zarówno w cytoplazmie jak i w innych organellach głównie w jądrze komórkowym. Powstają na drodze spontanicznej separacji faz ciecz‑ciecz (LLPS, ang. liquid-liquid phase separation) w odpowiedzi na zmieniające się czynniki fizykochemiczne mikrootoczenia. Składają się zarówno z białek które najczęściej posiadają regiony inherentnie nieuporządkowane (ang. intrinsically disordered regions, IDRs) jak i kwasów nukleinowych – głównie RNA. Niniejsza praca przedstawia informacje o biofizycznych podstawach formowania i funkcjonowania MLOs oraz zjawiska odpowiedzialnego za ich formowania, tj. LLPS. Zawiera także przegląd technik stosowanych w biochemii i biologii molekularnej, które za cel mają dostarczenie informacji o samoregulacji składu, struktury poszczególnych składników i ich lokalizacji oraz funkcjonalności danego MLO.

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