CHIP ubiquitin ligase is involved in the nucleolar stress management

The nucleolus is a dynamic nuclear biomolecular condensate involved in cellular stress response. Under proteotoxic stress, the nucleolus can store damaged proteins for refolding or degradation. HSP70 chaperone is a well-documented player in the recovery process of proteins accumulated in the nucleolus after heat shock. However, little is known about the involvement of the ubiquitin-proteasome system in the turnover of its nucleolar clients. Here we show that HSP70, independently of its ATPase activity, promotes migration of the CHIP (carboxyl terminus of HSC70-interacting protein) ubiquitin ligase into the granular component of the nucleolus, specifically after heat stress. We show that while in the nucleolus, CHIP retains mobility that depends on its ubiquitination activity. Furthermore, after prolonged exposure to heat stress, CHIP self-organizes into large, intra-nucleolar droplet-like structures whose size is determined by CHIP ubiquitination capacity. Using a heat-sensitive nucleolar protein luciferase, we show that excess CHIP impairs its regeneration, probably through deregulation of HSP70. Our results demonstrate a novel role for CHIP in managing nucleolar proteostasis in response to stress.

proteins after exposure to environmental stimuli or stress factors. For example, labile 49 proteins during heat stress are transported into the nucleolus, where the heat shock 50 protein 70 (HSP70) protects them from aggregation and facilitates their extraction and 51 refolding after stress (Nollen et al., 2001;Frottin et al., 2019). Thus, the HSP70 52 chaperone is essential for the maintenance of nucleolar proteostasis. Recent 53 proteomic analysis of the nucleolus from heat-shock treated cells identified numerous 54 proteins accumulating in nucleoli among which several belonged to the ubiquitin-55 proteasome system (UPS) (Azkanaz et al., 2019). UPS regulates various cellular 56 pathways by removing unwanted and damaged proteins marked by a small protein -57 ubiquitin (Ub). Its attachment is mediated by the Ub-activating enzymes (E1), Ub-58 conjugating enzymes (E2), and Ub ligases (E3) that select target proteins. In most 59 cases, the proteasome subsequently degrades ubiquitinated proteins (Komander,60 2009; Buetow and Huang, 2016). However, little is known about the involvement of the 61 UPS in nucleolar stress response and proteostasis maintenance. Recent studies 62 identified numerous proteins bound to NPM1 after heat shock (Frottin et al., 2019). 63 Their accumulation was transient, only under heat shock, and HSP70 activity was 64 required for their dissociation from NPM1 during recovery (Frottin et al., 2019). 65 Interestingly, several E3 ligases were detected in the aforementioned study, but further 66 investigation of their nucleolar functions was not carried out. One of these was the 67 quality control E3 ligase CHIP (C-terminus of Hsc70-interacting protein), the well-68 known HSP70 interactor. CHIP contains three tandem tetratricopeptide repeat (TPR) 69 motifs that bind to the HSP70 and HSP90 chaperones and the catalytic U-box domain 70 responsible for substrate ubiquitination (Ballinger et al., 1999;Jiang et al., 2001). Early 71 work showed that heat-treated CHIP retains its ubiquitination activity and can modify activation was significantly reduced. On the other hand, its turnover rate also 79 decreased, indicating that HSP70 and CHIP closely collaborate on degrading the 80 chaperone's substrates, and their interaction is also self-regulatory (Dai et al., 2003;81 Qian et al., 2006). However, it is unclear what is the role of CHIP while in the nucleolus 82 and whether it also cooperates with HSP70 in maintaining nucleolar proteostasis 83 during heat stress and recovery. 84 85 Here we show that heat shock-induced CHIP migration to nucleoli depends on HSP70 86 presence but not its activity. Nevertheless, functional HSP70 is essential for the 87 release of CHIP from the nucleolus. We also noted that nucleolar CHIP could exhibit 88 ubiquitination activity during heat stress and recovery. Specifically, CHIP is recruited 89 to the GC compartment where it acts as a non-aggregating protein; however, its 90 mobility becomes significantly limited when deprived of ubiquitination ability. 91 Remarkably, CHIP localizes to specific condensates generated in the nucleolus under 92 prolonged heat stress and whose dynamics depend on its E3 activity. To this end, we 93 used luciferase as a stress-sensitive model protein sorted to the nucleolus during heat 94 shock and observed that CHIP hinders its regeneration, likely in collaboration with 95 HSP70. Our results provide the groundwork for further studies on CHIP function in a 96 nucleolar heat stress response. 97 98 applied to inhibit HSP70 activity in HeLa cells during recovery from the 3 h heat shock 149 (Mediani et al., 2019). We observed CHIP levels gradually increasing in the nucleoli of 150 HeLa EGFP-CHIP and MCF7 cells during heat shock, implying that its nucleolar 151 migration was not affected by HSP70 inhibition (Fig. 2D and S1E). However, 152 continuous VER treatment during heat shock and recovery blocked CHIP release 153 during recovery, which resembled the effect of HSP70 depletion ( Fig. 2D and S1E). 154 These results suggest that HSP70 recruits CHIP in an activity-independent manner 155 upon entry to the nucleolus, but its functional operability in this compartment is required 156 for the recovery process and consequent CHIP release. When VER was provided only 157 during the recovery stage, CHIP clearance from nucleoli was only slightly reduced ( Fig.  158 2E), indicating that CHIP trapping in nucleoli depends primarily on the functionality of 159 the HSP70 during heat shock. 160 161 Therefore, we wanted to determine whether CHIP in the nucleolus acts as a functional 162 protein  194 reduction in size, and the formation of FBL nucleolar caps ( Fig. S3A and S3B).

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However, it did not induce CHIP migration to nucleoli ( Fig. 4A-C). These results support 196 the concept that CHIP is involved in the nucleolar heat stress response process rather 197 than, for example, suppressing rRNA transcription defects. While treatment with Act D 198 prior to heat shock did not affect CHIP migration to nucleoli ( Fig. 4B and 4C), it altered 199 CHIP distribution, which more prominently overlapped with Act D-induced NPM1 ring 200 formations (Fig. 4D). In addition, in cells exposed to Act D, CHIP exit from the nucleolus 201 during the 2 h heat stress recovery was partially impaired (Fig. 4B, 4C  ubiquitination activity (Fig. 5A). This is in line with the mobile and unaggregated 215 nucleolar fraction of CHIP ( Fig. 2F and G) and implies its capability of performing self-216 or substrates' ubiquitination. We also investigated whether CHIP activity is required for 217 its translocation using the catalytically-inactive CHIP H260Q mutant (Hatakeyama et 218 al., 2001). We found that the activity of CHIP is not indispensable for heat shock-219 induced migration to the nucleolus (Fig. 5B). However, FRAP analysis of the nucleolar 220 CHIP H260Q mutant showed a decrease in its dynamics compared to CHIP WT, 221 suggesting that its propensity to aggregate is likely mediated by the loss of 222 ubiquitination activity ( Fig. 5C and 5D translocates to nucleoli after heat shock and relocates to the nucleoplasm during 245 recovery. We verified a similar luciferase shuttle using our heat shock/recovery 246 scheme (Fig. 1A) and noted that transiently overexpressed CHIP (tagged with 247 mCherry) colocalizes with luciferase during heat shock (Fig. 6A). To investigate the 248 role of CHIP in nucleolar luciferase processing, we expressed its K30A and H260Q 249 mutants, which inhibit HSP70 binding or CHIP activity, respectively, in the 250 aforementioned HEK293T cell line. As a proxy for luciferase abundance and 251 regeneration, we analyzed the number of its foci in nucleoli during heat shock and the 252 6 h recovery period (Fig. S4A). Luciferase foci number decreased progressively during 253 the recovery, but in cells expressing specifically CHIP WT or CHIP H260Q, their 254 regeneration was slower than in untransfected and mCherry controls (Fig. S4A). 255 Notably, in cells expressing the CHIP H260Q mutant luciferase recovery was not 256 completed within the experimental 6 h time frame. This could be due to the high 257 number of cells containing heat shock-induced luciferase foci and their presence in 258 about 20% of non-heat shocked cells, suggesting that loss of CHIP activity had a potent 259 destabilizing impact on luciferase. Therefore, we decided to normalize our data to 260 correct for the differences in the number of luciferase foci during heat shock and control 261 conditions, focusing explicitly on the ability of CHIP variants to affect luciferase 262 nucleolar regeneration. Our analysis revealed that the elevated CHIP level induced a 263 delay in the dissolution of nucleolar luciferase foci during recovery (Fig. 6B).

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Overexpression of CHIP WT and CHIP H260Q had the most potent effect on reducing 265 luciferase exit from the nucleolus, and there was no difference in the rate of luciferase 266 recovery between the two variants. In contrast, overexpression of the CHIP K30A 267 mutant exerted a marginal effect on this process (Fig. 6B). When we transfected cells 268 with lower amounts of plasmids to induce milder overexpression of CHIP variants and 269 examined the first two hours of recovery from heat shock, we still observed comparable 270 inhibition of decline of luciferase foci during recovery by CHIP WT and the H260Q 271 mutant and no significant effect of CHIP K30A (Fig. S4B). We assumed that this was 272 due to the inefficient transport of CHIP K30A to nucleoli, as in HeLa Flp-In cells (Fig.  273  1E). Surprisingly, we found comparable redistribution of all CHIP variants to nucleoli 274 during heat shock, suggesting an alternative pathway for CHIP recruitment to nucleoli 275 unaccompanied by HSP70 in HEK293T cells (Fig. 6C). Hence, the above results 276 suggest that the slowed resolution of luciferase foci in nucleoli may be related to cross-277 talk between CHIP and HSP70.

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We next assessed luciferase levels as the ratio of its intensity between nucleolus and 280 nucleoplasm, as measured immediately after heat shock and during the 6 h recovery 281 period. The distribution of luciferase in untransfected or mCherry-transfected control 282 cells was predominant in the nucleoplasm already at the initial stage of recovery. 283 However, in cells overexpressing CHIP WT, the nucleolar luciferase signal was still 284 noticeable after 3 h of recovery, again indicating that the regeneration rate of luciferase 285 was disrupted (Fig. 6D). While the CHIP K30A mutant showed the least disruption in 286 the redistribution of luciferase, the CHIP H260Q mutant resulted in its most extended 287 nucleolar persistence (Fig. 6D). We also observed that CHIP was leaving the nucleoli 288 during recovery, concomitantly with nucleolar luciferase disappearance, with the 289 slowest rate for the CHIP H260Q mutant (Fig. 6C). Thus, we assume that CHIP-290 dependent ubiquitination may contribute to luciferase processing in nucleoli and 291 regeneration efficiency.

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As prolonged heat shock was shown to compromise nucleolar quality control and 294 inhibit luciferase regeneration (Frottin et al., 2019), we set out to investigate the effects 295 of CHIP on regeneration under these conditions. We measured luciferase intensity in 296 nuclei and nucleoli and monitored the number of luciferase foci during the 6 h heat 297 shock at 42°C. Control cells and cells expressing CHIP K30A, but not cells expressing 298 CHIP WT and H260Q, were capable of almost complete dissolution of luciferase foci 299 (Fig. S5A, S5B). However, we observed sustained sequestration of CHIP H260Q into 300 nucleoli after prolonged heat stress (Fig. S5C). Thus, we concluded that CHIP 301 repressed rather than enhanced nucleolar luciferase regeneration. Furthermore, our 302 results on CHIP K30A suggest that the interaction of CHIP with HSP70 may play a role 303 in modulating the nucleolus regeneration capacity and CHIP translocation to the 304 nucleoplasm. 305 306 HSP70 inhibition aggravates the negative effect of CHIP on luciferase 307 regeneration 308 309 We next examined the effect of CHIP on luciferase regeneration in the presence of 310 VER, the HSP70 inhibitor. Cells were treated with VER only during post-heat shock 311 recovery, and the number of nucleolar luciferase foci was measured after 1 h and 2 h 312 of recovery. In untransfected cells, we did not record any impact of VER on luciferase 313 regeneration. Cells overexpressing CHIP WT showed mildly impaired nucleolar 314 luciferase regeneration in the presence of VER, which became apparent after the 315 second hour of recovery compared to condition where it was absent (Fig. 7A). 316 However, overexpression of the CHIP K30A in cells with added VER had a more 317 disruptive effect relative to untreated cells (Fig. 7B). The result for the CHIP K30A 318 mutant was unexpected as, unlike the WT protein, it should not interfere with the 319 HSP70 function, which may suggest the emergence of additional effects associated 320 with the chaperone inhibition. Since the negative effect on luciferase regeneration in 321 CHIP H260Q-expressing cells was also potently enhanced by HSP70 inhibition, we 322 speculate that protection against protein aggregation in the nucleolus requires a 323 balance between HSP70 and E3 CHIP activity.
The nucleolus possesses numerous functions, including ribosome biogenesis, nuclear 328 organization, regulation of global gene expression, and energy metabolism (Cerqueira 329 and Lemos, 2019). It also responds to multiple stresses, such as hypoxia, pH 330 fluctuations, redox stress, DNA damage, or proteasome inhibition, and acts as a 331 protein quality control center that can mitigate heat shock-induced proteotoxicity 332 ( proteins in the nucleolus, we set out to study the protein quality control ubiquitin ligase 346 CHIP, which is well known for its role in ubiquitination of HSP70 substrates, and whose 347 presence in the nucleolus after heat stress has been reported in recent proteomic 348 showed that approximately 30% of total CHIP was immobile in the nucleolus in the 366 HeLa EGFP-CHIP cells. Heat shock also induces a similar formation of the immobile 367 GFP-NPM1 protein fraction, which implies altered properties of the GC due to its 368 association with misfolded proteins that accumulate in this phase upon heat shock 369 (Frottin et al., 2019). Thus, it is likely that CHIP embedded in GC associates with 370 aggregated proteins, which affects its mobility.

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What is the role of CHIP in the nucleolus? We hypothesized that in collaboration with 373 HSP70, CHIP might serve as a ubiquitin ligase or co-chaperone that regulates 374 ubiquitination or substrate reassembly to aid in the regeneration process. To revise 375 this, we focused on recovering a specifically modified luciferase that contained a 376 nucleus-targeting sequence to facilitate its accumulation in the nucleolus upon heat 377 shock. The effect of CHIP on luciferase status during heat shock and recovery but not 378 in association with the nucleolus was previously studied in vitro and in cellulo, showing 379 ambiguous results. CHIP can maintain denatured luciferase in a state capable of 380 folding and ubiquitinate it in vitro (Rosser et al., 2007). Moreover, heat shock may 381 enhance CHIP chaperone activity and its ability to suppress luciferase aggregation in 382 vitro (Rosser et al., 2007). In heat-stressed HEK293 cells, it was demonstrated that 383 CHIP overexpression protected luciferase activity and did not cause its increased 384 degradation. CHIP was also able to specifically interact with thermally denatured 385 luciferase rather than with the refolded one (Rosser et al., 2007). In fibroblasts, CHIP 386 overexpression did not affect luciferase degradation after heat shock and during 387 recovery but increased its HSP70-dependent reassembly and protected it from heat-388 induced insolubility (Kampinga et al., 2003 comparison test (****P < 0.0001, ***P < 0.001, **P < 0.01, ns P > 0.05). 499 E) HSP70 inhibition by VER during post-heat shock recovery only slightly affects 500 CHIP clearance from nucleoli. HeLa EGFP-CHIP cells were exposed to 90 min 501 heat shock and treated with 40 µM VER before transferring them for the 2 h-502 recovery. CHIP intensity was measured in nucleoli in control cells during heat 503 shock and recovery. Data are means of four independent experiments. Error 504 bars show SD. Statistical significance was determined using two-tailed unpaired 505 t-tests for pairwise comparisons (****P < 0.0001, ***P < 0.001, *P < 0.05 was determined using a one-way ANOVA followed by Dunnett's multiple 546 comparison tests (****P < 0.0001, *P < 0.05). 547 C) Pretreatment with 0.05 µg/ml Act D before heat shock does not affect CHIP 548 migration to nucleoli during heat shock but impairs its exit. Quantification of the 549 percentage of cells with CHIP present in nucleoli after 90 min heat shock and 550 2 h-recovery in HeLa EGFP-CHIP cells pretreated with Act D for 30 min or 2 h. 551 Data are means of three independent experiments. Error bars show SD. 552 Statistical significance was determined using a one-way ANOVA followed by 553 Dunnett's multiple comparison tests (**P < 0.01). 554 D) Treatment with Act D prior to heat shock alters CHIP distribution in nucleoli. 555 HeLa EGFP-CHIP cells were pretreated with 0.05 µg/ml Act D for 2 h before 556 heat shock, followed by immunostaining for NPM1 and confocal imaging. 557 Representative images and their magnified views of cells after heat shock (HS) 558 vs. cells treated with Act D before heat shock (Act D + HS) are shown. 559 Scale bars represent 10 µm or 5 µm (magnified views). 560 561 Fig. 5  Error bars represent SD. For statistical comparison a two-way ANOVA with post 645 hoc Tukey's test was used (***P < 0.001, **P < 0.01, *P < 0.05). 646 C) CHIP is redistributed to nucleoli during heat shock and leaves this compartment 647 during recovery. During recovery, the CHIP H260Q mutant's exit from nucleoli 648 is the slowest compared to CHIP WT and CHIP K30A. HEK293T cells 649 permanently expressing luciferase were transfected with vectors encoding for 650 mCherry-CHIP WT, mCherry-CHIP H260Q and mCherry-CHIP K30A and 651 treated with 2 h heat shock followed by 6 h recovery. Cells during treatments 652 were imaged live using confocal microscopy. Images were analyzed for the 653 mean mCherry intensities as a proxy for CHIP concentrations in the nucleoli and 654 nuclei, and the relative intensities were quantified. Data are means of three 655 independent experiments. Error bars represent SD. For statistical comparison 656 a two-way ANOVA with post hoc Tukey's test was used (**P < 0.01, ns P > 657 0.05). three independent experiments and are expressed as a % of total cell counts. 691 Error bars represent SD. For statistical comparison a two-way ANOVA followed 692 by Tukey's multiple comparison test was used (***P < 0.001, **P < 0.01, 693 *P < 0.05). Tukey's test was used ***P < 0.001, **P < 0.01, *P < 0.05). 785 786 Figure S5. HeLa EGFP-CHIP cells, nucleoli were manually selected, and the CHIP (EGFP) 978 intensity (mean gray value) was calculated. For colocalization studies, the JaCoP 979 plugin was used (Bolte and Cordelières, 2006). The image background was corrected 980 using the rolling ball algorithm (rolling ball=150). Thresholds of the green and red 981 channels were selected manually and maintained in every image.

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Analysis of the fluorescence intensity ratio -nucleolus: nucleus in HEK293T cells 984 985 Nucleoli were manually located using the Hoechst 33342 channel. Relative luciferase 986 or CHIP concentrations in nucleoli/nuclei were calculated based on GFP or mCherry 987 intensities, respectively, in each compartment in 50 cells per condition across three 988 biological repeats.