A Chemical Chaperone Decouples TDP-43 Disordered Domain

3 days ago - Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of str...
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A Chemical Chaperone Decouples TDP-43 Disordered Domain Phase Separation from Fibrillation Kyoung-Jae Choi, Phoebe S. Tsoi, Mahdi Muhammad Moosa, Adriana PaulucciHolthauzen, Shih-Chu Jeff Liao, Josephine C. Ferreon, and Allan Chris M. Ferreon Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01051 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Biochemistry

A Chemical Chaperone Decouples TDP-43 Disordered Domain Phase Separation from Fibrillation Kyoung-Jae Choi,§ Phoebe S. Tsoi,§ Mahdi Muhammad Moosa,§ Adriana Paulucci-Holthauzen,⊥ ShihChu Jeff Liao,‡ Josephine C. Ferreon,§,* Allan Chris M. Ferreon§,* AUTHOR ADDRESS. § Department of Pharmacology and Chemical Biology, Baylor College of Medicine, One Baylor Plaza, N520.03, Houston, TX 77030, USA ⊥ Department of Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA ‡ ISS, Inc., 1602 Newton Drive, Champaign, IL 61822, USA KEYWORDS. amyloid fibril, FLIM, osmolyte, prion-like domain, single-molecule FRET. Supporting Information Placeholder ABSTRACT: Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low complexity disordered domain of the archetypical SG-protein TDP-43 as model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggests that selective targeting of protein fibrillation (without altering condensation) can be employed as therapeutic strategy for RNP aggregation-associated degenerative disorders.

Stress granules (SGs) are dynamic RNA-protein assemblies that form upon cellular stress exposure1. They are membraneless organelles (MLOs) that enrich translationally inhibited ribonucleoproteins (RNPs) and act as sites for mRNA triage1. SGs are, however, double-edged swords; while indispensable in fending off a variety of cellular assaults, SG persistence is detrimental2-4. Indeed, genetic evidences implicate SGs as the key subcellular compartment involved in the pathogenesis of several RNP aggregation-linked degenerative disorders2, 3. The higher local concentration of aggregation-prone RNPs within SGs is assumed to facilitate pathologic protein transformation by aiding fibril nucleation and templating5-8. Recent reports indicate that ribonucleoprotein liquid-liquid phase separation (LLPS) drives the formation of SGs and other cellular RNP MLOs9. LLPS involves non-equilibrium homogeneous solutions separating into high- and low-density phases of unequal compositions5. Interestingly, many of the MLO component RNPs themselves undergo LLPS in vitro and form protein-dense droplets6-8. Such phase transitions are often driven by the low-complexity disordered regions (LCDs) of RNPs 6, which are usually prone to misfolding and aggregation10. Indeed, it was shown that LLPS facilitates aggregation of many phase separating protein systems presumably by protein enrichment6-8. Consequently, for many degenerative disorders, prevention of

LLPS-promoted pathologic fibrillation has been a key therapeutic strategy11, 12. Living systems employ various strategies to prevent pathologic protein transformations, including the use of chemical chaperones and osmolytes13. These small-molecule organic compounds function by perturbing the energetics of protein folding and ensembles14, 15. They are utilized to counteract the deleterious effects of extreme conditions such as high osmotic/hydrostatic pressures, dehydration, and high/low temperatures13. Among these chemical chaperones, trimethylamine N-oxide (TMAO) was shown to be highly effective in stabilizing protein native folds both in vitro and in vivo14, 16. Here, we study the effects of TMAO on LLPS-mediated RNP aggregation using the LCD of a representative SG-associated ribonucleoprotein, TDP-43.

Figure 1. The chemical chaperone trimethylamine N-oxide (TMAO) induces TDP-43LCD liquid-liquid phase separation (LLPS). (a) TDP-43LCD readily forms spherical droplets in the presence of TMAO. Droplet formation is enhanced with increasing TMAO concentration (a, top row). Condensation can

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be directly probed by monitoring changes in fluorophore fluorescence lifetimes (a, bottom row and b). (b) Fluorescence lifetimes are estimated by nonlinear least squares (NLS) fitting of the lifetime histograms to a Gaussian function (0 M TMAO, 3.71 ± 0.05 ns; 0.5 M TMAO, 2.83 ± 0.05 ns; 1 M TMAO, 2.81 ± 0.05 ns; and, 2 M TMAO, 2.78 ± 0.05 ns). Measurements were performed using 25 µM unlabeled TDP-43LCD and 1 nM Alexa Fluor 488-labeled protein in αβγ buffer. (c) In-droplet enrichment of TDP-43LCD is dependent on TMAO concentration. In-droplet (IN) and bulk (OUT) protein concentrations are presented as green and red bars, respectively. See Supplementary Methods for details. TDP-43 is composed of N-terminal oligomerization, two RRM, and C-terminal LCD domains17. The LCD domain harbors majority of TDP-43 disease-linked mutations and is required for TDP-43 SG recruitment18. Similar to LCDs of other SGassociated proteins (e.g., FUS, hnRNPA1, TIA1)6, 7, 18, TDP43LCD undergoes self-interaction-mediated LLPS19. Using fluorescence microscopy, we tested whether TMAO affects TDP43LCD LLPS (Figures 1, S1 and S3). At physiologic buffer conditions and in the absence of TMAO, 20-25 µM TDP-43LCD equilibrates as a homogeneous solution, absent of phase separation (Figures 1a, S1 and S3). In contrast, immediately after adding TMAO, we observed the formation of dynamic TDP43LCD-enriched droplets that fused to become progressively larger and settled at the bottom surface (Figure S3). To assess changes in protein solution dynamics, we employed fluorescence lifetime imaging microscopy (FLIM). FLIM is ultrasensitive to dye microenvironment changes and has recently been used to monitor protein aggregation20. In non-LLPS conditions, the fluorescence lifetime of Alexa 488-labeled TDP-43LCD is 3.56 ± 0.05 ns, which is not significantly perturbed by the presence of unlabeled protein (Figure 1b, top panel) or TMAO (Figure S4). However, upon TMAO-facilitated LLPS (with 20-25 µM unlabeled protein), the fluorescence lifetimes decrease dramatically to 2.7-2.8 ns (Figure 1b). The decrease in fluorophore lifetimes is likely due to indroplet local RNP enrichment (Figure 1c). The results of our fluorescence microscopy experiments clearly show that TMAO promotes LLPS of TDP-43LCD and enriches the protein within the droplets.

Figure 2. TMAO chaperoning prevents TDP-43LCD fibrillation. (a) Fluorescence microscopy images of TDP-43LCD with increasing TMAO concentration demonstrating the inhibitory effects of the chemical chaperone on aggregate formation (6-day incubation of samples presented in Figure 1). Spherical droplets morphed into irregularly shaped “starbursts” upon protein aggregation (0-1 M TMAO). At 2 M TMAO, droplets remained mostly spherical, suggesting fluid character. These spherical droplets were negative for Thioflavin T (ThT) binding (b; 3-day incubation; 20 µM unlabeled TDP-43LCD with 69 nM Alexa-594labeled protein in αβγ buffer). (c) Transmission electron microscopy images show the presence or absence of fibrillation at low or high TMAO concentrations, respectively. See Supplementary Methods for details. To directly test for the effects of TMAO on TDP-43LCD aggregation, we utilized a Thioflavin T (ThT) fluorescence assay and transmission electron microscopy (TEM). Without TMAO, TDP-43LCD formed aggregates within 3 days of room temperature incubation (non-LLPS conditions; Figure 2b). These aggregates formed directly from the homogeneous liquid phase and increased in their size upon prolonged incubation (Figures S2 and S3). TEM micrographs detected fiber-like aggregates (Figures 2c and S5), providing direct evidence for fibrillation. We also observed concomitant fluorophore lifetime decrease to 2.63 ± 0.05 ns (not shown) as TDP-43LCD aggregated, suggesting that FLIM probes both protein condensation and aggregation. In the presence of TMAO, however, we observed a TMAO concentration-dependent reduction of protein fibrillation despite the chemical chaperone promoting LLPS (Figures 2, S2 and S3). After prolonged incubation, droplets that formed in the presence of 2 M TMAO remained mostly spherical (Figures 2, S2 and S3). At 2 M TMAO, after 11 days of room temperature incubation, the presence of aggregates was undetected even with the highly enriched indroplet TDP-43LCD concentration (~184 µM ; Figures S2 and S5). Overall, our results suggest that in phase separating conditions, TMAO facilitates TDP-43LCD LLPS yet inhibits protein fibrillation in a concentration-dependent fashion. To decipher the molecular basis of TMAO action on protein phase separation and fibrillation, we utilized the distance dependence of single-molecule Förster/fluorescence resonance energy transfer (smFRET) to directly visualize LCD conformations. For smFRET experiments, TDP-43LCD was dyelabeled at residues 310 and 368, encompassing the ‘amyloidogenic’ core 21. We recorded fluorescence bursts from freely diffusing dye-labeled proteins using a custom-built microscopy setup. Donor and acceptor signals were analyzed to generate smFRET histograms, allowing direct visualization of protein conformational distributions. The smFRET histogram of the protein alone exhibited a single non-zero EFRET peak at 0.77 ± 0.01 (Figure 3a). Zero-peaks in smFRET histograms originate from molecules lacking active acceptor dyes. To check whether TDP-43LCD is disordered, we performed smFRET guanidine hydrochloride (GdnHCl) denaturation titration. With increasing [GdnHCl], we observed a non-cooperative expansion of the LCD as evidenced by the non-sigmoidal decrease in measured EFRET values (Figure 3b). The absence of a cooperative unfolding transition in the denaturation experiment provides evidence for the protein’s disorderedness22. Conversely, we observed a gradual protein compaction in isothermal smFRET TMAO titration (Figure 3). A non-cooperative transition along with the absence of more than one non-zero EFRET peaks in our titration data suggest that TMAO shifts TDP-43LCD structural ensembles towards more collapsed forms without inducing specific conformations.

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Biochemistry Unlike their marginally stable counterparts, natively unfolded disordered proteins lack complete folding code in their primary sequence and are therefore unable to minimize the unfavorable protein backbone-TMAO interactions through population of compact folded conformations15, 24. Instead, these proteins can minimize backbone exposure to TMAO by accessing higherbarrier but energetically favored aggregated states25-27. Our data, however, suggests that LLPS provides an alternate strategy for minimizing the unfavorable TMAO interaction with the protein backbone (i.e., rather than fold or aggregate to states of minimal backbone exposure, proteins can escape to phase separated compartments; Figure 4). We observe that TMAO enriches TDP43LCD within the condensates, minimizing TMAO exposure to the disordered LCD backbone by progressive protein depletion from the low-density phase (L1 in Figure 4, bottom panel). A likely outcome of such protein depletion is the significant reduction of protein aggregation from the low-protein density phase as evidenced by the markedly decreased amount of inter-connected starburst-like assemblies with increasing TMAO (Figure 2). Counterintuitively, our results also demonstrate that TDP-43LCD aggregation is clearly disfavored from the high-protein density phase (L2 in Figure 4, bottom panel). Despite nearly 35x proteins enrichment at 2 M TMAO, TDP-43LCD droplets remained ThTnegative upon extended incubation (Figures S2 and S3). Overall, we observe that protein aggregation is disfavored from both the high- and low-protein density phases in the presence of sufficient concentrations of the chemical chaperone.

Figure 3. TMAO forced compaction of the disordered TDP-43 low-complexity domain. (a) TDP-43LCD is structurally disordered as evidenced by a non-cooperative denaturant-induced expansion (in contrast to the cooperative unfolding expected for folded proteins). TMAO compacts the LCD ensemble in a similarly noncooperative fashion. Zero-peaks are shaded gray. (b) Mean EFRET plotted as functions of cosolvent concentrations. EFRET values were obtained by NLS fitting of individual smFRET histograms to a Gaussian function. Solid lines are provided as guides. From a mechanistic perspective, TMAO is preferentially excluded from the protein backbones due to the unfavorable interaction energetics14. For disordered proteins at sufficiently low protein concentrations, TMAO preferential exclusion results in compact protein conformations22, 23. Accordingly, we observed TMAO-forced compaction of TDP-43LCD with increasing concentrations of the chemical chaperone in the protein dilute phase (Figure 3). The molecular picture for the high-protein density phase is, however, less clear. Compared to the proteindilute phase: (1) the protein-dense phase has less TMAO molecules per TDP-43LCD due to the significant in-droplet protein enrichment (TMAO concentrations were approximately the same in both phases; Figure S6 and Supplementary Methods); (2) interprotein interactions are enhanced within the protein-dense phase; and (3) the solution environment is significantly different in the protein-rich phase (e.g., similar TMAO concentration in both phases and increased protein concentration in the demixed phase should result in reduced water content in the high-density phase). All these factors are likely to contribute differentially to in-droplet TDP-43LCD monomer conformations.

Figure 4. Decoupling of TDP-43 liquid-liquid phase separation (LLPS) from fibrillation using TMAO. TMAO facilitates LLPS of TDP-43LCD and inhibits fibrillation in a concentration-dependent fashion (top panel). Bottom panel presents a schematic representation of the energetics of TMAO-induced LCD phase transitions. Left: Unfavorable TMAO-polypeptide backbone interaction energetics induces LLPS where TDP-43LCD in-droplet (L2 phase) enrichment minimizes protein backbone cosolvent exposure; direct fibrillation from this protein-enriched L2 phase is disfavored. Right: Phase diagram of TDP-43LCD homogeneous liquid, liquid demixed and fibrillar states illustrating how protein and TMAO concentrations can modulate phase transitions. Low-complexity domains of many ribonucleoproteins are linked to both physiologic condensation and pathologic fibrillation18, 28. A recent report by Eisenberg and coworkers suggested that segments within the TDP-43LCD are responsible for the protein’s reversible assembly and irreversible fibrillation21. Our results clearly show that the chemical chaperone TMAO promotes TDP-43LCD phase separation yet disfavors fibrillation.

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Our observed anti-correlated effects of TMAO on condensation and fibrillation of an archetypical RNP-LCD suggests that these two processes are not necessarily coupled. Decoupling of ribonucleoprotein physiologic phase separation from their pathologic fibrillation presents a viable therapeutic approach for SG protein-linked degenerative disorders.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supplementary figures, supplementary methods and supplementary references (PDF)

AUTHOR INFORMATION Corresponding Authors * Email: [email protected]. * Email: [email protected]. Author Contributions J.C.F. and A.C.F. designed the experiments. K.J.C. prepared the TDP-43 DNA constructs and performed most of the fluorescence microscopy imaging experiments, and along with J.C.F. collected and analyzed the NMR data. A.P.H. collected some of the microscopy images. J.C.F. purified and labeled protein samples. P.S.T. and A.C.F. collected and analyzed the smFRET data. J.C.F. and A.C.F. collected and analyzed the FLIM data, with assistance from S.C.J.L. J.C.F., M.M.M., and A.C.F. wrote the manuscript. Funding Sources This work was supported by laboratory startup funds from the Baylor College of Medicine (A.C.F. and J.C.F.). P.S.T. was supported by a fellowship (F31 NS103380) from the NINDS, NIH. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank Debra Townley from the Baylor College of Medicine Microscopy Core for the TEM data collection.

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Biochemistry

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