scholarly journals Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

2019 ◽  
Author(s):  
Amandeep Girdhar ◽  
Vidhya Bharathi ◽  
Vikas Ramyagya Tiwari ◽  
Suman Abhishek ◽  
Usha Saraswat Mahawar ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Binding Site ◽  
Liquid Phase Separation ◽  
Rna Molecules ◽  
Mechanism Of Inhibition ◽  
Rna Depletion ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractTDP-43 is an RNA/DNA-binding protein of versatile physiological functions and it is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease in addition to several other implicated proteins such as mutant SOD1 and FUS etc. Cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation of TDP-43 are suggested to be important TDP-43-toxicity causing mechanisms for the ALS manifestation. So far, therapeutic options for ALS are extremely minimal and ineffective therefore, multi-faceted approaches such as treating the oxidative stress and inhibiting the TDP-43’s aggregation are being actively pursued. In our recent study, an acridine imidazolium derivative compound, AIM4, has been identified to have anti-TDP-43 aggregation propensity however, its mechanism of inhibition is not deciphered. In this study, we have utilized computational methods to examine binding site(s) of AIM4 in the TDP-43 structure and have also compared its binding efficiency with several other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic core region of amino acids aa: 288-319, which coincides with one of the key residue motifs that could potentially mediate liquid-liquid phase separation (LLPS) of TDP-43. Importantly, alike to the previously reported effects exerted by RNA molecules, we found that AIM4 could also inhibit the in vitro LLPS of a recombinantly purified C-terminal fragment TDP-432C bearing an A315T familial mutation. Antagonistic effects of AIM4 towards LLPS which is believed as the precursor process to the TDP-43’s aggregation and the in silico prediction of a binding site of AIM4 on TDP-43 occurring in the same region, assert that AIM4 could be an important molecule for further investigations on TDP-43’s anti-aggregation effects with relevance to the ALS pathogenesis.

scholarly journals TDP-43 α-helical structure tunes liquid-liquid phase separation and function

2019 ◽  
Author(s):  
Alexander E. Conicella ◽  
Gregory L. Dignon ◽  
Gül H. Zerze ◽  
Hermann Broder Schmidt ◽  
Alexandra M. D’Ordine ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Changes ◽  
Helical Structure ◽  
Liquid Phase Separation ◽  
Gene Assay ◽  
And Function ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. Present in several MLOs, TDP-43 undergoes LLPS and is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some disease variants of TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes in a dimeric TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a mini-gene assay. Therefore, TDP-43 helical region serves as a short but uniquely tunable module that shows promise as for controlling assembly and function in cellular and synthetic biology applications of LLPS.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Liquid–Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 548
Author(s):  
Donya Pakravan ◽  
Emiel Michiels ◽  
Anna Bratek-Skicki ◽  
Mathias De Decker ◽  
Joris Van Lindt ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Hydrophobic Interactions ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
End Stage ◽  
Positive Effect ◽  
Self Aggregation ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

scholarly journals Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

2022 ◽  
Author(s):  
Ewa Niedzialkowska ◽  
Tan M Truong ◽  
Luke A Eldredge ◽  
Stefanie Redemann ◽  
Denis Chretien ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Chromosome Segregation ◽  
Dynamic Structure ◽  
Liquid Phase Separation ◽  
Chromosomal Passenger Complex ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Liquid Liquid Phase Separation

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

scholarly journals Liquid–Liquid Phase Separation in Crowded Environments

2020 ◽  
Vol 21 (16) ◽  
pp. 5908 ◽  
Author(s):  
Alain A. M. André ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Macromolecular Crowding ◽  
Liquid Phase Separation ◽  
The Impact ◽  
Crowded Environments ◽  
Liquid Liquid Phase Separation ◽  
In Cells

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

scholarly journals Granulins modulate liquid–liquid phase separation and aggregation of the prion-like C-terminal domain of the neurodegeneration-associated protein TDP-43

2020 ◽  
Vol 295 (8) ◽  
pp. 2506-2519 ◽  
Author(s):  
Anukool A. Bhopatkar ◽  
Vladimir N. Uversky ◽  
Vijayaraghavan Rangachari
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Disorder ◽  
Liquid Phase Separation ◽  
Proteolytic Processing ◽  
Full Length ◽  
Cytoplasmic Inclusions ◽  
Terminal Domain ◽  
Liquid Liquid Phase Separation

TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hallmarks of both FTLD and ALS are the toxic cytoplasmic inclusions of the prion-like C-terminal fragments of TDP-43 CTD (TDP-43 C-terminal domain), formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. Both full-length TDP-43 and its CTD are also known to form stress granules by coacervating with RNA in the cytoplasm during stress and may be involved in these pathologies. Furthermore, mutations in the PGRN gene, leading to haploinsufficiency and diminished function of progranulin (PGRN) protein, are strongly linked to FTLD and ALS. Recent reports have indicated that proteolytic processing of PGRN to smaller protein modules called granulins (GRNs) contributes to FTLD and ALS progression, with specific GRNs exacerbating TDP-43–induced cytotoxicity. Here we investigated the interactions between the proteolytic products of both TDP-43 and PGRN. Based on structural disorder and charge distributions, we hypothesized that GRN-3 and GRN-5 could interact with the TDP-43 CTD. We show that, under both reducing and oxidizing conditions, GRN-3 and GRN-5 interact with and differentially modulate TDP-43 CTD aggregation and/or liquid–liquid phase separation in vitro. GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid–liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS.

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 Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

2021 ◽  
Author(s):  
Nazanin Farahi ◽  
Tamas Lazar ◽  
Shoshana J. Wodak ◽  
Peter Tompa ◽  
Rita Pancsa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Liquid Liquid Phase Separation ◽  
In Cells ◽  
Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

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