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Widespread remodeling of proteome solubility in response to different stresses

Xiaojing Suia, Douglas E. V. Piresa,b,c, Angelique R. Ormsbya, Dezerae Coxa, Shuai Nied, Giulia Vecchie, Michele Vendruscoloe, David B. Aschera, Gavin E. Reida,f,1, and Danny M. Hattersa,1

aDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC 3010, Australia; bCentro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais 30.190-009, Brazil; cSchool of Computing and Information Systems, The University of Melbourne, VIC 3010, Australia; dMelbourne and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, VIC 3010, Australia; eDepartment of Chemistry, Centre for Misfolding Diseases, University of Cambridge, CB2 1EW Cambridge, United Kingdom; and fSchool of Chemistry, The University of Melbourne, VIC 3010, Australia

Edited by Jason Gestwicki, University of California, San Francisco, CA, and accepted by Editorial Board Member William F. DeGrado December 20, 2019 (received for review July 29, 2019) The accumulation of protein deposits in neurodegenerative the protein homeostasis system (6, 7). Studies employing Cae- diseases has been hypothesized to depend on a metastable norhabditis elegans models have shown that the aggregation of subproteome vulnerable to aggregation. To investigate this polyQ can cause temperature-sensitive mutant proteins, phenomenon and the mechanisms that regulate it, we mea- which are metastable in their native states, to aggregate (7), sured the solubility of the proteome in the mouse Neuro2a which is consistent with a reduced capacity of the quality control line under six different protein homeostasis stresses: 1) system. Similarly, in aging, different genetic backgrounds and Huntington’s disease proteotoxicity, 2) , 3) , 4) pro- environmental stresses can alter the efficiency of the protein teasome, 5) (ER)-mediated folding inhi- homeostasis system (8, 9). bition, and 6) . Overall, we found that about Here, we sought to address three outstanding questions re- one-fifth of the proteome changed solubility with almost all lated to the factors capable of compromising protein homeo- of the increases in insolubility were counteracted by increases stasis. The first question is how does aggregation of mutant in solubility of other proteins. Each stress directed a highly spe- Httex1, which previously has been suggested to unbalance pro- cific pattern of change, which reflected the remodeling of protein tein homeostasis, impact the aggregation state of the wider complexes involved in adaptation to perturbation, most notably, proteome? The second is which proteins in the proteome are stress granule (SG) proteins, which responded differently to differ- metastable to aggregation under different triggers of protein ent stresses. These results indicate that the protein homeostasis homeostasis stress? And the third is whether there is a sub- system is organized in a modular manner and aggregation pat- terns were not correlated with stability (ΔG). In- proteome that consistently requires a functional protein ho- stead, distinct cellular mechanisms regulate assembly patterns of meostasis machinery to remain folded and soluble, and if so, multiple classes of protein complexes under different stress how is this subproteome regulated? Our results indicate that a conditions. substantial proportion of proteome undergoes large changes, both upward and downward, in solubility in response to stress, protein homeostasis | protein aggregation | protein misfolding | Huntington’s disease | molecular chaperones Significance

he protein homeostasis system is vital for cell function as it The protein homeostasis system prevents proteins from mis- Tensures that proteins are properly translated, folded, traf- folding and aggregation. It is unclear, however, how this sys- ficked to their correct cellular locations, and eventually degraded tem is organized to fulfil this role under different insults. To in a tightly controlled and timely manner. As a major task for this investigate this problem, we examined how six different system is to prevent damaged and misfolded proteins from ac- stresses affected proteome solubility in a mouse neuroblas- cumulating, it has been hypothesized that, when this system toma cell line. We found that, although different stresses becomes unbalanced, proteins become prone to misfolding, substantially changed the solubility of many proteins, no pro- which results in their mislocalization and deposition as aggre- teins substantially aggregated in common. Instead, each stress gates (1, 2). Indeed, dysfunctional protein aggregation and pro- distinctly remodeled protein–ligand networks involved in key tein homeostasis imbalance are central pathological features of cellular processes, notably SG formation and . We common neurodegenerative diseases including Alzheimer’s conclude that cells are endowed with a wide range of regula- (AD), Parkinson’s (PD), Huntington’s, and motor dis- tory responses, which are tailored for distinct stresses and eases (3, 4). buffer against widespread protein aggregation. Different neurodegenerative diseases are characterized by the presence of signature proteins in characteristic deposits formed Author contributions: X.S., D.C., S.N., G.E.R., and D.M.H. designed research; X.S., A.R.O., and S.N. performed research; X.S., D.E.V.P., A.R.O., D.C., G.V., M.V., D.B.A., G.E.R., in the brains of affected patients. These proteins include TDP-43 and D.M.H. analyzed data; and X.S. and D.M.H. wrote the paper. and FUS in motor neuron disease, τ and Aβ in AD, α-synuclein ’ The authors declare no competing interest. in PD, and huntingtin (Htt) in Huntington s disease. It has been This article is a PNAS Direct Submission. J.G. is a guest editor invited by the Editorial suggested that these aggregation-prone proteins, which may be Board. affected by (e.g., Htt), posttranslational modifications Published under the PNAS license. τ (e.g., ), or environmental changes, lead to Data deposition: The mass spectrometry proteomics data have been deposited in the oversubscription of quality control resources that overloads the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identi- protein homeostasis system (5). This imbalance then triggers a fiers (accession nos. PXD014420 and PXD015573). cascade of protein folding defects that broadly impairs proteome 1To whom correspondence may be addressed. Email: [email protected]. function. In cell models expressing mutant Htt 1 (Httex1), This article contains supporting information online at https://www.pnas.org/lookup/suppl/ for example, key molecular systems are sequestered doi:10.1073/pnas.1912897117/-/DCSupplemental. into inclusions formed by Httex1, which depletes the resources of First published January 21, 2020.

2422–2431 | PNAS | February 4, 2020 | vol. 117 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1912897117 Downloaded by guest on September 24, 2021 but also that each stress is associated with an articulated stress protein and for proteins that show large proportional changes in response that affects a different part of the metastable sub- solubility. On the other hand, pellet ratios are expected to proteome. Our data further suggest that the majority of the be selectively sensitive for proteins that have very small fractions changes arises from the functional remodeling of protein– of aggregate or proteins that are less abundant in the proteome. ligand complexes in adaptation (or response) to the stress and Hereon, we use the term solubility to indicate the changes in that the changes are highly specific to the different stress protein mass measured by this specific experimental framework. factors. Based on these results, we conclude that the resilience Our first protein homeostasis stress model examined the effect of the protein homeostasis system against widespread aggrega- of Httex1 and the aggregation state on the overall tion is based on the absence of a large common set of metastable proteome solubility. Huntington’s disease mutations lead to the proteins that aggregate under multiple stresses. Furthermore, the expansion of a polyglutamine (polyQ) sequence in Httex1 to proteins that change aggregation do not correlate with the protein lengths longer than 36Q, whereas the wild-type protein is typically folding stability (ΔG). In this view, the proteins that change sol- less than 25Q (11). PolyQ expansion causes Httex1 to become ubility most are functionally responsive to protein homeostasis highly aggregation prone, which manifests as intracellular in- regulators that are organized into different modules that respond clusion bodies as the disease progresses (12). We and others have specifically to different perturbations. used transient expression constructs of Httex1 fused to fluorescent proteins as models for replicating essential features of the disease, Results including protein homeostasis stress (13, 14). Mutant Httex1- Httex1 Mutation and Subsequent Aggregation Distinctly Remodels fluorescent protein constructs progressively form large cytosolic Proteome Solubility. To investigate how protein homeostasis inclusions in cell culture over time. imbalance alters the aggregation state of the proteome, we We employed the flow cytometry method of pulse shape employed a neuronal-like cell model system (mouse Neuro2a analysis to purify cells expressing mutant Httex1-mCherry into cells) and a quantitative proteomic workflow inspired by the those with inclusions (i) from those without inclusions ([ni]; dis- work of Wallace et al. (10). In essence, the approach involved a persed uniformly in the ) at matched median expression fractionation strategy based on centrifugation of cell lysates levels (15). This strategy enabled us to assess how the aggregation prepared using a mild nonionic detergent based lysis condi- state of mutant Httex1 (97Q ni and i) affected proteome solubility — tion (IGEPAL CA-630) with subsequent quantitative proteome compared to a wild-type state (25Q ni note that 25Q does not analysis to monitor changes in the abundances of individual form aggregates) (Fig. 2A and SI Appendix, Fig. S1). proteins between the supernatant versus the pellet, resulting First, we assessed the protein abundances using the Experi- BIOCHEMISTRY from each stress (Fig. 1). Quantitation was performed using a ment 1 pipeline from Fig. 1A. A total of 2,013 proteins were reductive dimethylation labeling approach with n = 4 biological identified (Dataset S1). Only a handful of them (22) significantly changed abundance among the 25Q-ni, 97Q-ni, and 97Q-i sam- replicates and detection by nano-reversed-phase liquid chroma- – tography coupled with tandem mass spectrometry (MS/MS). We ples (Fig. 2B). None of these had known protein protein inter- measured the changes in the abundance of proteins in the total actions (Fig. 2C), and the dataset was enriched with only one GO term ( GO:0005856, Dataset S2). However, several starting material (Experiment 1 in Fig. 1A) and applied two ’ reported methods to measure changes in solubility that provide of the proteins have reported roles in Huntington s disease- complementary information (10). Δ pSup was defined as the relevant mechanisms. Further discussion of the functional de- SI Appendix change in proportion of the protein in the supernatant by sub- tails of these proteins is in , Note 1. For the assessment of protein solubility changes, we observed tracting the proportion of the protein in the stress (pSup ) Stress 2,519 proteins* (Fig. 2B). For the comparison between the 97Q-ni from the supernatant of control (pSup ) (Experiments 2 and Control and the wild-type 25Q-ni, we observed a slightly higher proportion 3 in Fig. 1B). We also measured the changes in the pellet fraction of proteins that decreased solubility in 97Q-ni (17 more soluble directly as the ratio of proteins in the stress:control treatments and 22 less soluble). Likewise, a slightly higher proportion of (Experiment 4 in Fig. 1B). The measure of pSup is expected to be proteins became more insoluble once Httex1 formed inclusions the best estimate of change in the absolute yield of soluble (97Q-ni versus 97Q-i) (16 more soluble and 25 less soluble). None of these differences were statistically significant (χ2 test P = 0.89; Dataset S3—test 1). Control Stress Control Stress Of the proteins that significantly changed solubility due to ABpellet pellet Total dispersed or inclusion-localized 97Q Httex1, almost one-third Total lysate 100,000g 100,000g lysate (24 out of 78) were previously reported as interactors of Httex1, 20min 20min five of which became more soluble (16) and 19 of which becaame In-solution digestion more insoluble (16–20) (Fig. 2C, shown in bold). The enrichment dimethyl labeling, mix 1:1 Supernatant Pellet Pellet Supernatant of known Httex1 interactors was not significant (Fisher’s exact test P = 0.36; Dataset S3—test 2). One interesting feature was

Trypsin digestion, reductive dimethylation, mix 1:1, mass spec that 11 of the Httex1-interacting proteins were reported to lo- calize to SGs including Cttn, Pds5b, Cpsf3, Ddx3x, Dnajc7, pSup =S/T Pellet ratio=P/P pSup – Intensity Control Stress=S/T Eif4a3, Ubqln2, Nup88, Pcbp1, Fus, and Srsf10 (21 26). Eight (Stress/Control) m/z other SG proteins were also observed to change in solubility, T P T S including Helz2, Mthfd1, Serbp1, Eif5a, Eif4b, Cdv3, Pdap1, and P S Data analysis Flnb. The enrichment of SG proteins to the proteins that Intensity Intensity Intensity m/z m/z m/z changed solubility was statistically significant overall and within Experiment 1 ’ = = Experiment 2Experiment 4 Experiment 3 the Httex1 interactors (Fisher s exact test P 0.0009 and P — Outputs in two complementary measures of solubility changes: 0.02; Dataset S3 tests 3 and 4). Further examination of two of pSup pSup pSup 1. Change in ( Control – Stress) these SG proteins (Eif4a3 and Fus) by (Fig. 2 2. Change in insoluble fraction (Pellet ratio, stress:control) D–I) indicated subtle changes in their localization. Eif4a3 resided Fig. 1. Proteomic workflow for quantitative measurements of stress-related proteome solubility changes. (A) Strategy to measure changes in total pro- teome abundances due to stress (Experiment 1). (B) Strategies to measure *This includes the sum of proteins seen in the 25Q-ni versus 97Q-ni and 97Q-ni versus 97Qi changes in solubility by a combination of experiments (Experiments 2–4). treatments.

Sui et al. PNAS | February 4, 2020 | vol. 117 | no. 5 | 2423 Downloaded by guest on September 24, 2021 Downloaded by guest on September 24, 2021 foewyAOAadTukey and ANOVA one-way of er2 el rnfce ihHtx-Cer n muotie ihFs hehlsanttda ecie for described as annotated Thresholds Fus. ( structures. in with Data ring cell. immunostained the per to and spots corresponds of Httex1-mCherry that size threshold Average with brightness transfected defined a cells above Neuro2a immunofluorescence Eif4a3 nuclear of Proportion te1mhryadimnsandwt i43 hehlsfrncerrn tutrs(e atr)adncesbudr wiedse ie)aeshown. are lines) dashed (white boundary nucleus and pattern) (red structures ring nuclear for Thresholds ( Eif4a3. with immunostained and Httex1-mCherry eeotlg G)trsaeanttd l nihdG em r nlddin included are terms GO enriched All annotated. are terms (GO) ontology and 1 of onsidct rtisblwtetrsodo infcne(wfl hneand change (twofold significance of threshold the below proteins indicate points 2424 cells denotes ni shown. as levels expression matched with PulSA by groups ( three inclusions into with presorted cells were denotes (97Q) i polyQ and mutant inclusions, and without (25Q) polyQ wild-type for 2. Fig. C DE A E (non-inclusions) (non-inclusions) (non-inclusions) (non-inclusions) itgaso i43imnfursec nest nietencesadi h yols a es,nn el eeaaye o ahhsorm.( histogram). each for analyzed were cells nine least, (at the in and nucleus the inside intensity immunofluorescence Eif4a3 of Histograms ) (inclusions) (inclusions) Eif4a3 | 25Q Thresholded Httex1 Eif4a3 97Q 97Q 97Q 97Q 25Q mato te1mtto n usqetageaino ouiiyo noeospoen.( proteins. endogenous of solubility on aggregation subsequent and mutation Httex1 a of Impact www.pnas.org/cgi/doi/10.1073/pnas.1912897117 P au f00) ( 0.05). of value 25Q Down-regulated B –log (p-value) –log (p-value) 0 1 2 3 4 0 2 4 6 Down-regulated Down-regulated -8-4048 Trim16 Gtf2b Lasp1 Tbce Ttc9b b11 Epb4.1l3 Tbc1d15 Sympk Plk1 Gtf2b Ociad1 in 97Q-ni in 97Q-ni 6-4 -6 Trim16 Abundance 97Q C Log Plk1 rti ewrsrpeetdb TIG(.10 nlsswt eimcniec neatos eetdsgiiatyenriched significantly Selected interactions. confidence medium with analysis (v.11.0) STRING by represented networks Protein ) 707 0 -7 2 ’ (foldchange) Rbm42 Tnks1bp1 Usp19 utpecmaiosts.* test. comparisons multiple s Nes Epb4.1l3 Msh2 Timm13 Ociad1 Marcks Lasp1 Abundance Aak1 Log Log F -20246 F 2 2 , (odchange) (fold change) (fold H 4 Kif5c Density (×10 ) Chaperone-mediated proteinfolding(GO:0061077) Rock2

and , Proportion of Rabgap1 Up-regulated A 10 15 20 lexaFluor 488fluorescence(AFU) nuclear Nop16 Lgmn 0 5 0040 6000 4000 2000 0 Nop16

fluorecence above Msh2 I Kif5c Timm13 l hwdtpit svle rmidvda el n ihmeans with and cells individual from values as datapoints show all Usp19

threshold Nes 0.00 0.10 0.20 Tbce Up-regulated Up-regulated in 97Q-ni in 97Q-ni B Rbm42 ocn lt fpoenaudne n ouiiy lge oteshmtcin schematic the to aligned solubility, and abundances protein of plots Volcano ) 5 7-i97Q-i 97Q-ni 25Q Emd Nup88 Dnajb1 St13 Fus Psap Necap1 Tkt * P < Bold =Httex1interactors 97Q-i nuc 97Q-i cyto 97Q-ni nuc 97Q-ni cyto 25Q nuc 25Q cyto 0 1 2 3 4 5 0 1 2 3 4 5 .5ad**** and 0.05 10-. . . 1.0 0.5 0.0 -0.5 -1.0 1.0 0.5 0.0 -0.5 -1.0 Gm6563 Mtap7d2 Hbs1l Ktn1 Tmpo Snrpa Dnaja2 Dnajc7 Cttn Arhgdib Hbs1l Vps29 oesoluble More soluble More in 97Q-ni in 97Q-ni Serbp1 Pcnp Cfdp1 Cpsf3 Sub1 Uchl3 Pds5b Pds5b Csnk1d Eif5a Serbp1 P P Supernatant Eif5 GH Scamp3 < au f00 o bnac and abundance for 0.05 of value Eif4a3 aae S2 Dataset Fus .01 Saebr,10 bars, (Scale 0.0001. Spots Httex1 Fus Ddx3x Srsf10 pSup pSup Grpel1 Srrm2 Pmpcb Mthfd1l Tpd52l2 Cttn Srsf10 25Q Dnajb1 essoluble Less soluble Less .( ERAD pathway(GO:0036503) in 97Q-ni in 97Q-ni St13 Hist2h2bb Cfdp1 Hadh Rab1b Tmpo D Scamp3 Ubqln2 ofclmcorpso er2 el rnfce with transfected cells Neuro2a of micrographs Confocal ) Solubility Pin4 Bag6 Sgta Sgta Pdap1 Eif4b Pnp Rsu1 97Q Solubility Cops3 Sgta A μm.) er2 el rninl xrsigHttex1-GFP expressing transiently cells Neuro2a ) Ubqln1 Ubqln2 A Cth Hgs Pcbp1 ldoa 0 1 2 3 4 5 0 1 2 3 4 8 8- 8 4 0 -4 -8 -4 esinsolubleLess insolubleLess I Tamm41 ± in 97Q-ni in 97Q-ni Vps29 Eif5a Δ More soluble Dson hs aesas hwresults show also panels These shown. SD Dbnl Helz2 pSup Des Gatad2a Des Average spot Eif5 Eif4a3 Hist1h2bb Number of Lamtor1

D area per cell Hist2h2bb

.( spots per cell au f03 eltrtolog ratio Pellet 0.3, of value

(pixels) 200 400 600 800 H13 Rfc1 Tamm41 10 15 H BC006779 0 0 5 Log Log 0 ubro pt e el ( cell. per spots of Number ) Dync1li1 Dbt Nmt2 Maf 2 2 G 101 0 -1 Pellet (odchange) (fold change) (fold Rab3a ofclmcorpsof micrographs Confocal ) 5 7-i97Q-i 97Q-ni 25Q 5 7-i97Q-i 97Q-ni 25Q ∆ pSup Pcbp1 f Lmnb2 Rab1b **** Ddx3x Fus Uqcrfs1 Lrrc40 Flnb Picalm 4 Cdv3 More insoluble A **** oeinsoluble More insoluble More A tp6v0a1 Gray-colored . in 97Q-ni in 97Q-ni Ptcd3 Sgta Hgs Pin4 Tkt Ubqln1 Pds5b A Pnp Rsu1 Eif5a Pcnp u tal. et Sui A Bag6 rhgdib Eif5a 2 rhgdia value 8 F I ) ) predominately in the nucleus and was enriched in punctate struc- Fig. S2B). Similar to what was observed for protein abundance tures within the nucleus similar to what has been previously de- changes, the GO terms indicated functional groupings anticipated scribed (27) (Fig. 2D). The 97Q treatments led to reduced nuclear from protein functions related to the treatment (Fig. 3B for three staining of Eif4a3 (Fig. 2E) and, in particular, within the ringlike examples of Hsp90 inhibition by novobiocin treatment, protea- structures (Fig. 2F). Fus also resided mostly in the nucleus but also some inhibition, and oxidative stress; SI Appendix,Fig.S3for the formed cytoplasmic puncta as anticipated for SGs (Fig. 2G). The other two of Hsp70 inhibition and ER stress). This result, there- 97Q treatment did not appear to change the number of Fus puncta fore, indicates that the protein assembly state, not just abundance, (Fig. 2H), but it did increase the size of the puncta (Fig. 2I). is key to understanding the function of the protein homeostasis Of note is the broader role for SG abnormalities involved with function (Fig. 3B). For example, MG132 treatment indicated en- – misfolded proteins and neurodegenerative diseases (28 30). A richment for (GO: 0006508) as anticipated. An effect GO analysis revealed 37 terms enriched in the proteins that on activity was also indicated by MG132 increasing changed their solubility when mutant Httex1 97Q formed in- the abundance of and proteasome subunits (SI Appen- clusions. This set of terms included chaperone-mediated protein dix,Fig.S4A). Almost all of the proteolysis GO terms were folding (GO:0061077) and ER-associated protein degradation associated with proteins becoming more insoluble, suggesting pathway (GO:0036503) (full list of enriched GO terms in Dataset that the proteasome-degradation machinery forms larger molecular S2). Collectively, these data suggested that mutant Httex1 causes two weight complexes when the proteasome is inhibited, which is con- major effects on the proteome. The first is a substantial remodeling sistent with the prior knowledge that proteasome inhibition induces of SG proteins into different cellular locations both before and after the formation of ubiquitin- and proteasome-enriched cellular aggregation into inclusions, which includes some elements becom- aggregates (43) (Fig. 3B). Further insight into this functional ing more soluble and some less soluble. The second is that the quality control systems involved in ER stress and protein misfolding remodeling of the proteasome machinery was provided by the appear to be selectively remodeled to become less soluble as Httex1 substantial decrease in solubility of total cellular ubiquitin, even inclusions form, which is consistent with the inclusions recruiting though an overall increase in total ubiquitin abundance was molecular chaperones and other quality control machinery in at- observed (SI Appendix,Fig.S4B). tempts to clear them (6, 16). Another notable finding from this analysis was the ability to extract novel information on the effect of novobiocin treatment Different Triggers of Protein Homeostasis Stress Invoke Distinct on assembly states of macromolecular machines. Novobiocin did Functional Remodelings of Proteome Solubility. To investigate not change the levels of Hsp90 or proteins involved in the heat

whether the proteins that changed solubility upon Httex1 aggre- shock response as anticipated but decreased known Hsp90 client BIOCHEMISTRY gation are relevant to protein homeostasis stress more generally, proteins, in accordance with previous studies (Fig. 3A) (44, 45). we expanded our analysis to examine proteome solubility changes GO and network analysis of the changes in solubility identified associated with five other triggers of protein homeostasis stress many more Hsp90 clients than those detected from expression that have previously reported roles leading to protein misfolding level analysis as well as large changes in the solubility of proteins and aggregation. These stresses included three specific inhibitors in diverse complexes including those that form the proteasome, of key protein homeostasis hubs (Hsp70, Hsp90, and the protea- mitochondrial , DNA repair machinery, RNA splicing some) whereby defects are reported in models of Huntington’s machinery, RNA transport machinery, and respiratory chain disease, protein aggregation, degradation of misfolded proteins, complexes (Fig. 3B). Despite these substantial changes in pro- and/or other markers of protein homeostasis stress (6, 16, 31) and teome solubility, the enrichment of Hsp90 clients was not sig- two exogenous stress states that reflect observed in nificant (Fisher’s exact test P = 0.32 and P = 0.75; Dataset S3— neurodegenerative disease settings and protein aggregation in cell tests 5 and 6), which may be a consequence of off-target impacts models (namely, oxidative stress and ER stress) (32–35). We driving much of the changes in the proteome since novobiocin chose approaches that could be readily and relatively specifically has an IC50 value around 700 μM (46). To explore this idea targeted pharmacologically and that have been well studied pre- further, we tested a different Hsp90 inhibitor, 17-allylamino-17- viously to cause protein homeostasis stress. demethoxy-geldanamycin (17-AAG) (EC50 of 7.2 μM) that The Hsp70 chaperone system was targeted by the small molecule operates through a distinct mechanism by inhibiting ATPase inhibitor Ver-155008, which binds to the ATPase domain of Hsp70 μ – μ function through binding to its amino-terminal domain (47). family proteins (Kd of 0.3 MandIC50 of 0.5 2.6 M) (36, 37) and Unlike novobiocin, inhibition by this mechanism is known to can promote the aggregation of a reporter metastable protein in activate the heat shock response (48). In accordance with this transfected cells (38). Hsp90 was targeted with the ATP binding effect, 17-AAG increased Hspa8 and other competitor novobiocin, which can unbalance the protein homeo- proteins in the protein folding GO term (GO: GO:0006457) stasis system without activating a compensatory heat shock re- (Fig. 4). Like that for the novobiocin treatment, we observed a sponse and induce the aggregation of a metastable bait protein (38). Proteasome activity was targeted with the inhibitor MG132 substantial change in solubility (upward and downward) of a wide range of proteins, including a statistical enrichment of (39). ER stress was triggered using the N-linked glycosylation in- ’ = — hibitor tunicamycin, which impairs flux of folding via the calnexin– Hsp90 client proteins (Fisher s exact test P 0.02; Dataset S3 calreticulin folding pipeline (40). Oxidative stress was induced with test 7) and proteins in the protein folding GO term. There was a arsenite (41, 42). Our experimental design followed the dosages limited overlap in proteins that changed solubility with no- and timings as performed in prior studies as indicated above. vobiocin (shown in Dataset S5). The most notable difference The changes in protein abundance from these treatments are between the treatments was that novobiocin appeared to impair shown in SI Appendix, Fig. S2A (the full proteomics datasets are some complexes from properly assembling into large molecular summarized in Datasets S4–S9). The treatments led to changes weight machines, including the mitochondrial respiratory chain, consistent with their function based on GO assignment as well as which contains five multimeric membrane-anchored complexes protein–protein interactions (Fig. 3A; complete list of GO terms (49). We observed more than half of identified subunits of mi- in Dataset S2). Of note was that many more proteins were ob- tochondrial respiratory complexes I, III, and IV becoming more served to have changed solubility (upwards and downward) than soluble after novobiocin treatment, suggesting a failure of these had changed abundance, which suggests that protein solubility complexes to assemble into their mature states as part of large change, rather than changes in protein expression, is a particu- membrane-anchored complexes, which are anticipated to parti- larly substantial response to stress (volcano plots in SI Appendix, tion into the insoluble fraction under our pelleting regime.

Sui et al. PNAS | February 4, 2020 | vol. 117 | no. 5 | 2425 Downloaded by guest on September 24, 2021 AB

Fig. 3. Impact of three protein homeostasis stresses on proteome abundance and solubility. Protein–protein interaction analysis of proteins with significant changes in abundance (A) or solubility (B) due to protein homeostasis stresses (Hsp90 inhibition [with novobiocin], proteasome inhibition [with MG132], and oxidative stress [with arsenite], top view) was performed with built-in String (v.11) in Cytoscape (v3.7.1) at the medium confidence. Selected significantly enriched GO terms are annotated. All enriched GO terms are included in Dataset S2. Hsp90 interactions were manually added based on String and shown with thicker black connectors. SG curated list from Markmiller et al. (22).

† Consensus Features of Metastable Subproteomes Changing Solubility after pelleting . There was a small but significant decrease in under Stress. To address the fundamental question of whether solubility of 3.6% (P = 0.0047 by two-way ANOVA; Dataset S3) there is a common metastable subproteome that is more (Fig. 5A). These results suggest that aggregation arising from aggregation prone under any condition of stress due to pro- tein misfolding, we next sought to assess whether stress in- † creased the net insolubility of the proteome by measuring the Note we were unable to measure solubility for the Huntington’s disease cell model due protein mass concentration in the supernatants before versus to small yields of cells after flow cytometry sorting.

2426 | www.pnas.org/cgi/doi/10.1073/pnas.1912897117 Sui et al. Downloaded by guest on September 24, 2021 A Abundance Solubility Supernatant Pellet 10 8 8 Phf6 Fads1 8 Asf1b 6 Dnajc3 6 Ncor2 Myo1b Racgap1 Fyttd 6 Diexf Ccnk Hspa8 Csk Cdk1 4 Lypla1 4 Hsph1 Tpd52l2 Tor1aip1 Hsp90aa1 4 Luc7l2 Mapre2 –log(p–value) Sncg Atp6v1d Taf9 Chtf8 Pum1 2 Cycs 2 Stip1 2 Glul Acyp1 Ldlr 0 0 0 –8 –4 0 4 8 –1.0 –0.5 0.0 0.5 1.0 –8 –4 0 4 8 Log (fold change) pSup Log (fold change) Down–regulated 2 Up–regulated More soluble Less soluble Less insoluble 2 More insoluble in stress in stress in stress in stress in stress in stress pSup Log2 (fold change) ∆ B Down–regulated Up–regulated More soluble More insoluble –7 0 7 , DNA templated (GO:0006351) –1 0 1

Creb1 Gtf2e2 Phox2a Ascc3 Slc25a33 Adrm1 Racgap1 Ube2c Smc5 Ccnk Incenp Anapc7 Rcc2 Dctn6 Cul7 Ncapg2 Asf1b Baz2a Setd3 Polr1c Asz1 Cdca8 Kifc1 Ilk Dnmt3a Bccip Hjurp Trrap Foxk1 Gtf2h2 Hint1 Ak6 Taf5 Ash2l Cdk2ap1 Kifc1 Snx18 Arpp19 Mbd3 Nelfa Ikbkap Ssu72 Ahctf1 Mapre2 Zfp207 Esco2 Pcnp Tubgcp2 Haus5 Tubgcp3 Isl1 Gtf3c1 Ctnnd1 Ruvbl2 Dhx36 Cnot11 Tacc3 Ints3 Kif11 Tcea1 Rbpj Polr1a Eed Taf10 Mdc1 Snx18 Ppp2r2a Blm Cdc27 Tpx2 Eif4e Myh9 Setd3 Elp2 Edf1 Khsrp Cnot3 Phf6 Cdk7 Ilk Ncapd2 Pelo Trp53bp1 Prkaa1 Pelp1 Utp15 Tpx2 Aurkb Ints3 Kif4 Ncapd2 Nedd1 Birc6 Ndc1 Polr1b Asf1b Leo1 Polr1d Tfb1m Ascc1 Rnf2 Kif4 Ccnb1 Elp2 Cd3eap Xab2 Gtf2f2 Lig1 Prkar1a Orc3 Pds5a Ccnb1 Espl1 Ncapd3 Hells Myh10 Elp3 Morf4l2 Paxbp1 Trps1 Ncor2 Zcchc9 D2Wsu81ePpp1r12aTsg101 (GO:0007049) Adar Phox2a Atad2 Nucks1 Hells Myh10 Kif2c Kif22 Kif23 Smc5 Kif20b Racgap1Mdc1 Whsc1 Tceb2 Polr1a Ctr9 Polrmt Flii Slc25a33 C1qbp Rcc2 Anapc7 Ckap5 Tubg1 Dnmt1 Kdm1a Mta1 Sltm Uhrf1 Cdk11b Gsk3b Csnk1a1 Csnk1a1 Uhrf1 Camk2g Mre11a Dnmt1 Kdm1a Sp1 Cul4a Mlkl Rabggta Ogt Cdk1 Cdk4 Plk1 Gsk3b Pcna Plk1 Nsl1 Cdk1 Ranbp9 Prkci Hax1

Chordc1 Hspa8 Hsp90 clients Hspa8 Stub1 Fkbp8 Fam3c Obsl1 Lypla1 Psmg4 Edc4 Hspa5 Dnaja1 et al Mrps25 Trim47 Zyx Ndufc2 Cd63 Hsp90b1 (Taipale , 2012) Pdia6 Hsp90ab1 Fkbp1a Shcbp1 Eif4a1 Cttn Picalm Eif4h Prps1 Kif5c Hsph1 Stau1 Spag9 Cr1l Trmt6 Epb4.1l1 Ahsa1 Pdcl3 Xpnpep3Acyp1 Tpm4 Ldlr Cttnbp2nlEpb4.1l1Lypla1 Frmd4a Pdcd10 Ndufa2 Cnpy4 Szrd1 Dnajb1 Dnaja2 Dnajb1 Hsp90aa1 Hsph1 Lmnb1 Ddb1 Decr1 Plekhh3Abcf2 Acad9 Stip1 Spata5 Ociad1 Stbd1 Fads1 Gemin5 Protein folding Tpm1 Cnn3 Tomm70aWdr76 Gid8 Mogs Obsl1 Sdf2l1 Rab3a Map2k2 Prrc2c Clint1 BIOCHEMISTRY (GO:0006457) Tubb4a Map1a Prune2 Lamtor5Prpsap1Agpat5 Zfp330 Ubxn1 Ddx20 Rnh1 Metap2 Capn6 Wdr3 Dimt1 Utp14a Utp18 Crnkl1 Prkaca Exosc8 Ddx39 Luc7l3 Utp6 Sipa1l2 Nup210 Armc8 Ubr5 Ubap2 Mon2 Tbl2 Cpsf1 Manf Eefsec U2af2 Pold1 Sbds Rasl2–9 Pwp2 Rrp7a Wdr33 Ddx39b Wdr3 Fcf1 Trmt2a Utp11l Rap1b Hbs1l Capn6 Coro2b Plod1 Acot7 Acsl3 Sympk Uba6 Sparc Bcas2 Tpd52l2 Cpsf1 Dcaf13 Cpsf3 Nsun5 Nol6 Dimt1 Gch1 Slc25a10Aspscr1Ftl1 Dock7 Cox5a Twf1 Coro1b Mtx2 Mogs Gna13 Fth1 Wdr74 Tbl3 Diexf Utp6 RNA processing Ndufa10Rps19bp1Arpc1b Fscn1 Vps29 Ppp2r5a Myo5a Sez6l2 Impact Lrrc47 Apmap Luc7l2 Rbm26 Tbl3 Isy1 Ints1 Ddx51 Tor1aip1 Gnl2 Dcaf13 Rsl24d1 Lsm12 Ipo5 Bzw2 Kpna2 Cyb5b Pcx Map6 Tnpo3 Dkk3 M6pr Degs1 (GO:0006396) Ddx28 Tomm22 Tnks1bp1Arhgef1Csnk1e Actg1 Ythdf2 Myo1b Elmo2 Prepl Txndc9 Nup107 Uap1 Mpc2 Ccdc115 Dnajc11 Mrpl20 Coro1c Cplx2 Tbc1d31Atp1a1 Atp6v1dWdr1 Pus1 Ap2a1 Fam207aKpna2 Lancl1 Pum1 Mpst Thoc2 Des Agfg1 Ribosome Pfkp Fyttd1 Sipa1l1 Nup85 Actb Mapk8ip3 Mkln1 Aen Nucb2 Dnajc3 Heatr3 Ddx55 Timm50Ube2n Klc2 Pdlim7 Sncg Dhrs1 Ncdn Tor1aip1 Xpnpep3 Fam96b Scrib Prim1 biogenesis Rhox5 Ppa1 Mov10 Lsm14b Eef2 Tssc4 Irf2bpl Parvb Galnt2 Mrpl9 Eif4g3 Esyt1 Pcbp4 Rab2a Eif4g1 Nlrp4c Slc7a1 Flnb Hmmr Tmod3 Dock4 Hspa5 Nup133 Mccc1 Ep400 Lgmn Myo1c Lmnb2 Dhx33 Pnkp Ccdc137 (GO:0042254) Map6 Impdh1 Impdh2 Zfp598 Rftn1 Api5 Eif4g2 Actr10 Slc1a5 Rbm4 Tubb2bTagln2 Mcm10 Cycs Trmt5 Ipo8 Idi1 Nes Blvrb Uqcrh Hat1 Stom Stk39 Rangrf Vdac3 Emd Cad Sec61a1 Farsb Mpg Fth1 Nmd3 Acaca Carhsp1Tamm41Mrpl27 Degs1 Ubl4 Macf1 Rfc2 Abcf2 Igsf8 L2hgdh Mapk8ip3Esd Crat Glul Arhgef1 Qsox2 Sssca1 Rbx1 Fam203a Crip2 Psme4 Eif4g1 Akap1 Nle1 Fam207aCul4a Psmg3 Letmd1 Tmed10 Dhx29 Csk Cmas Ppp1r7 Tpm2 Ndufa6 Asrgl1 D10Jhu81eGch1 Ptges2 Glg1 Metap2 Srp54a Dnajc16 Luzp1 Rdx Orc2 Palm Gse1 Suclg1 Ndufs2 Lmtk2 Fbxo30 Ide Smchd1 Prepl Ufsp2 Fads2 Cd9 Plaa Ppfibp1 Prpf3 Col4a3bpCpsf2 Lrrc47 Pno1 Lrch3 Eif5 Tex30 Acat2 Surf4 Maged1Samm50Cfl2 Nlrp4c Myl9 Gspt1 Mrpl54 Peg10 Rnf126 Fam162aLrrc40 Rsu1 Chga Vamp7 Dock7 Dhx29 Pfkm Uap1 Mpst Pacsin2 Ccdc6 Acadvl Apobec3 Coro1b Eml4 Hint2 Cand2 Clint1 Sdcbp

Fig. 4. Impact of Hsp90 inhibition by 17-AAG on proteome abundance and solubility. (A) Shown are effects of 17-AGG treatment on abundance and sol- ubility. (B) Protein:protein interaction analysis of proteins with significant changes in abundance (Left) or solubility (Right) due to indicated protein ho- meostasis stresses was performed using built-in String (v.11.0) in Cytoscape (v3.7.1) at the medium confidence. Selected significantly enriched GO terms are annotated. All enriched GO terms are included in Dataset S2.

misfolded proteins increases marginally under stress but does observed that only 26 proteins became more insoluble in, at not reflect a dramatic accumulation of misfolded protein states least, three stresses, which represent just 0.6% of the proteome that aggregate. detected (Fig. 5B) while 139 proteins (2.6% of the proteins) At the individual protein level, there were no proteins that became more insoluble in, at least, two stresses. Only three consistently decreased or increased in solubility across six stresses proteins, Pcbp1, Bag3, and Sqstm, were more insoluble in four using our criteria for a significant change. Of the proteins detected stresses, and only one protein, asparagine synthetase (Asns), was in all experiments, 408 proteins significantly decreased solubility more insoluble in five of the stresses (Dataset S9). Likewise, for by any one or more of the stress treatments, which represent proteins that became more soluble, we observed only a low 10.5% of the proteins detected (4,278). In addition, 315 proteins number of proteins (8) that became more soluble in, at least, had significantly increased solubility (8.2%). A further 183 pro- three of the stresses (0.2% of the proteome detected) (Fig. 5B teins increased or decreased solubility depending on the stress and Dataset S9). No protein was found to be more soluble in (4.9%). Altogether, the proteins that changed solubility was just more than three stresses. Expanding the analysis to two stresses over one-fifth of the proteome (21.2%) with a significant bias to yielded 73 proteins (1.7% of the proteome). proteins becoming more insoluble (based on 95% confidence in- GO analysis of the proteins that changed solubility in two or tervals [CIs] on proportions; Dataset S3). Collectively, these data more stresses illuminated the key mechanisms involved (Fig. indicate that significant remodeling in proteome solubility occurs 6A). The most enriched molecular function terms for the more under stress but that most of the increase in insoluble protein load insoluble protein list were proteasome-activating ATPase activity is counterbalanced by other proteins increasing in solubility. (82.6-fold enrichment), proteasome binding (36.7-fold), struc- Despite this widespread change in solubility, the changes were tural constituent of nuclear pore (35.9-fold), ATPase activator largely specific to the type of stress invoked. For example, we activity, (9.9-fold), and heat shock protein binding (9.8-fold)

Sui et al. PNAS | February 4, 2020 | vol. 117 | no. 5 | 2427 Downloaded by guest on September 24, 2021 Control A 100 B Stress # proteins 80 More insoluble More soluble

60 Common in 591 498 at least 1 stress 40 Common in 139 73 in supernatant at least 2 stresses 20 % protein of amount Common in 26 8 0 at least 3 stresses 2 n enite 4278 rs Background MG13 A Novobiocin Ver-155008 Tunicamyci C More insoluble More soluble 97Q-ni vs 97Q-ni vs 97Q-i vs 97Q-i vs 25Q-ni 17 25Q-ni 15 21 97Q-ni 97Q-ni 2 1 8 23 1 2 1 1 1 2 Hsp70 2 Hsp70 2 inhibition 1 inhibition Oxidative 30 Oxidative 34 18 3 1 1 7 stress 101 1 2 stress 1 11 1 42 9 5 1 5 2 1 2 6 1 3 6 1 2 4 2 1 1 140 Hsp90 1 1 Hsp90 2 1 4 5 214 1 1 1 1 inhibition inhibition 54 10 1 1 22 43 7 1 11 3 8 4 (novobiocin) 5 4 3 (novobiocin) ER stress 89 54 ER stress Proteasome Proteasome inhibition Purple bold, SG proteins inhibition Cellular response to stress Nucleocytoplasmic transport (GO: 0033552) (GO: 0006913) Sumo2 Asns Psmd14 Kpna2 Eif5a Pcbp1 Cdv3 Rps21

Lmnb2 Polr2b Trim28 Pds5b Xpo5 Anp32e Ubap2l Mrps36 Tmpo Armc6 Anp32b Sqstm1 Bag6 Fscn1 Txn1 Flnc Cops3 Bag3 Aacs Aldh1l2 Mlf2 Vdac2 P4ha2 Atp5mg Pdap1

Fig. 5. Dynamic remodeling of proteome solubility involving a core enrichment of proteins involved in nucleocytoplasmic transport and SGs. (A) Overall proteome solubility changes due to treatments. Comparison of the proportion of protein amount in the supernatant fraction out of the total lysate measured by bicinchoninic acid assay between the control and the treatment groups. Error bars represent SD. N = 3or4.(B) List of proteins found in common as shown. (C) Venn diagram presents the overlaps of more insoluble or soluble proteins across six stresses. The area enclosed by the thick black line represents the overlap regions with, at least, three stresses. Protein–protein interaction of common proteins that became more insoluble (red) and more soluble (blue) in, at least, three stresses are shown. Purple bold represents known SG proteins.

ubiquitin protein ligase binding (6.0-fold). These functions are was the profound enrichment of SG proteins in both proteins consistent with protein quality control mechanisms being en- that become more insoluble as well as those that become more gaged to respond to proteome folding stress. Other major bi- soluble in, at least, three stresses (Fig. 5C). Indeed, there was a ological process and cellular component terms of enrichment significant enrichment of proteins involved in SGs that become include filament (99.0-fold), nuclear pore outer ring (66- both more insoluble and more soluble across one or more fold), mRNA export from the nucleus (22.0-fold), and protein stresses (Fig. 6B). Curiously, a majority of the SG proteins dis- import into the nucleus (14.6-fold), which are consistent with played differentially altered solubility depending on the stress previously reported findings that protein folding stress, more (Fig. 6C). These observations indicate a great diversity, dyna- generally, impacts nucleocytoplasmic transport mechanisms (24, mism, and heterogeneity in the complexes that are formed by SG 50–55). For the proteins that become more soluble, we observed proteins and are suggestive of an elaborate tailoring of the terms highly enriched for mitochondrial activity including ATPase functional responses of the SG structures to the stress. activity, coupled to the transmembrane movement of ions, rota- tional mechanism (46.9-fold), proton-exporting ATPase activity Discussion (40.0-fold), proton transmembrane transporter activity (15.5-fold), In this paper, we found that about one-fifth of the proteome of ATP biosynthetic process (21.8-fold), and mitochondrial protein mouse neuroblastoma cells undergoes solubility changes in re- complex (9.7-fold). sponse to an array of stresses to the protein homeostasis system. Another striking observation was that 183 of the proteins that We found that most of the proteins that decrease in solubility become more insoluble in one or more of the stresses also be- are counteracted by other proteins that increase in solubility. came more soluble in one or more of the other stresses (Dataset Different stresses, therefore, evoke a regulatory response that S9). This finding suggests that these proteins are functionally rebalances homeostasis that involves a large functional remod- tailored to different roles in different stresses. Clues to the key eling of protein–ligand interactions. It is also apparent that pathways involved came from examination of the proteins that protein aggregation arising from the perturbation of protein became more insoluble in, at least, three stresses, revealing GO quality control under these conditions accounts for only a minor enrichments for nucleocytoplasmic transport and cellular re- fraction of the changes in the proteome solubility (probably less sponse to stress, which are consistent with stress and prior than 5%). Indeed, if marginally stable proteins mediate the shift findings that nuclear-cytoplasmic transport is altered in neuro- in aggregation under protein homeostasis stress, we might pre- degenerative disease settings (Fig. 5C) (50–54). Another finding dict there to be a correlation between the free energy of folding

2428 | www.pnas.org/cgi/doi/10.1073/pnas.1912897117 Sui et al. Downloaded by guest on September 24, 2021 ∆ pSup A C More soluble More insoluble GO enrichment for Biological Process, More insoluble -1 0 1 RNA export from nucleus Pcbp1 Pdap1 protein import into nucleus Ubap2l Kpna2 Txn Bag3 ubiquitin-dependent protein catabolic pro... Ddx6 Fscn1 nucleic acid metabolic process Pds5b Nono cellular response to stress Cdv3 Eif5a Prrc2a Larp4 01234 5 Csde1 Flnb Log (fold enrichment) Sfpq Dnaja1 2 Atxn2l Hnrnpm GO enrichment for Cellular Component, More insoluble Polr2b Nup98 lamin filament Upf1 Ipo7 Cnot1 Rfc3 nuclear pore outer ring Pcna Anp32e proteasome regulatory particle Fus Ddx3x Ubap2 Rangap1 cytoplasmic ribonucleoprotein granule Map4 Eif4b nuclear membrane Serbp1 Fubp1 Eif3h Vasp inner membrane Nup88 Etf1 mitochondrial part G3bp1 Hspd1 cytosol Npm1 G3bp2 Stip1 Ewsr1 0 2468 Atp5f1a Anxa6 Log (fold enrichment) Gspt1 Ywhaq 2 Tpt1 Prdx1 GO enrichment for Molecular Function, More insoluble Lmna Pdia3 Rcc1 Rbbp4 proteasome binding Cdk1 Sf3a3 structural constituent of nuclear pore Pycr1 Nup93 ATPase activator activity Lin28a Rad23b heat shock protein binding Celf1 Ccar1 ubiquitin protein ligase binding Nup155 Nsun2 Actr1a Pcbp2 RNA binding Hnrnpk Coro1c adenyl ribonucleotide binding Rhoa Strap Metap1 Rfc4 024 6 Cttn Cpsf3 Log (fold enrichment) Pdlim5 Akap12 2 Dnajc7 Ybx3 GO enrichment for Biological Process, More soluble Zc3h14 Clint1 ATP biosynthetic process Eif2s2 Clpp proton transmembrane transport Spats2 Ybx1 Srsf3 Ddx19a 012345 Prdx4 Chchd3 Pfn2 Cnbp Log (fold enrichment) Phb2 Ywhab BIOCHEMISTRY 2 Pcmt1 P4hb GO enrichment for Cellular Component, More soluble Prmt5 Ranbp1 Rbm3 Hsp90aa1 Eif3c Vcp proton-transporting two-sector ATPase com... Nup50 Hspa4 sheath Eif3a Dpysl2 Ywhah Eif3l mitochondrial Cnn3 Prmt1 ribonucleoprotein complex Dynll2 Ubl4a Mcm5 Usp10 organelle envelope Gnb2 Rae1 cytosol Mcm4 Caprin1 intracellular non-membrane-bounded org... Smu1 Mcm7 Eif4g2 Eif3g 0 2468 Nup62 Uba1 Hnrnph2 Log (fold enrichment) Ik 2 Nop58 Fbl H1f0 Fxr1 GO enrichment for Molecular Function, More soluble Khsrp Tmod3 Usp5 Psmd2 ATPase activity, coupled to transm... Zc3hav1 proton-exporting ATPase activity Pfn1 Cap1 Ntmt1 proton transmembrane transporter activity Arpc1b Gfpt1 RNA binding Ddx21 Pura Rnh1 Tnpo1 0246U2af1 Tomm34 Log (fold enrichment) Ctnnd1 Ptges3 2 Edc4 Tcea1 B Kpnb1 Dpysl3 Snd1 Tubb3 1.0 More insoluble Hnrnpul1 Hspa9 Srp9 More soluble ** Chordc1 0.8 Ago2 Xpo1 Racgap1 Tufm Smc4 0.6 * **** Fmr1 FAM120A Mov10 Ppme1 Rcc2 0.4 Itgb1 proteins * ** Eif3k 0.2 Aldh18a1 0

Proportion of proteins ER stress that are stress granule Background 1+ 2+ 3+ 97Q-i vs97Q-niHsp70 inhibition Oxidative stress 97Q-ni vs 25Q-ni Hsp90 (novo)Proteasome inhib. inhib.

Fig. 6. Biological pathways involved in the functional remodeling of proteome solubility. (A) GO terms for proteins significantly changed in solubility in two or more stresses. Shown are the top tier GO terms tested with Fisher’s exact test and Bonferroni post hoc testing with P < 0.05. (B) The proportion of detected SG proteins using the curated list of Markmiller et al. (22) for proteins in the indicated number of stresses. Black circle indicates background proteome. Colored circles indicate proteins that significantly changed solubility. Fisher’s exact test results comparing stress to background are shown (*P < 0.05; **P < 0.01; ****P < 0.0001) (Dataset S3—test 9). Mean and 95% CIs shown from proportions analysis (Dataset S3)(C) Barcode graph of SG proteins from Markmiller et al. (22) and solubility.

(ΔGF) and solubility. Analysis of the proteome ΔGF values from candidate proteins, such as TDP-43 or τ which are commonly ref. 56 for the proteins in our dataset revealed no correlations for mislocalized and/or misfolded in neurodegenerative disease any individual stress or the pooled data (SI Appendix, Fig. S5 and contexts (57, 58). Instead, changes are the assembly state of Dataset S10). Our results, thus, reveal an important principle proteins that are dominated by functional responses to dif- underlying the robustness of the protein homeostasis system, ferent stresses (an alternative analysis of the core proteins that namely, that there is no bellwether set of proteins that are pro- change solubility to the data in Fig. 5C is shown in SI Ap- foundly metastable due to being difficult to fold, including obvious pendix,Fig.S6).

Sui et al. PNAS | February 4, 2020 | vol. 117 | no. 5 | 2429 Downloaded by guest on September 24, 2021 These findings are informative to a large-scale proteomic The protein seen in five stresses, Asns, has been reported to study of mouse models of different neurodegenerative diseases, form filaments in yeast under stress (65), suggesting it also is including AD, PD, and amyotrophic lateral sclerosis (59). In that functionally remodeled in the stress response. When we consider study, 91 proteins were found to become more sodium dodecyl the solubility changes in proteins found in, at least, three stresses sulfate (SDS) insoluble in three transgenic mouse models. Our in terms of KEGG pathways, we observed a clustering into core data show a significant enrichment for these proteins when we metabolic pathways of metabolism, metabo- considered proteins that decreased solubility by, at least, one lism, metabolism, metabolism, and energy stress (Fisher’s exact test P =<0.0001; Datasets S9 and S3–test metabolism (SI Appendix, Fig. S7). The clusters included pro- 10). These findings point to the general principles of proteome teins that increased and decreased solubility and suggest that the solubility remodeling operating as found in the mouse neuro- remodeling of the proteome solubility is functionally linked to blastoma cell context also in more chronic disease settings. core responses associated with the protein homeostasis stress In our dataset, there was only one protein, C (Flnc), induction. One last noteworthy point is that involved in that appeared to stand out as a potential bona fide bellwether metabolism pathways, including specifically those found in our protein for substantial aggregation by misfolding. Flnc was ob- paper as hotspots for changes in solubility, have been shown to served to become more insoluble in three stresses (ER stress, form molecular condensates from phase separation in yeast and oxidative stress, and proteasome inhibition). Mutations in Flnc other cell models (66), which suggests that functional aggrega- cause myofibrillar , which are characterized by pro- tion changes by phase separation could drive many of the func- tein deposits, a defective ubiquitin–proteasome system, and ox- tional changes seen in the data presented here, such as SGs. idative stress (60). Flnc is a cytoskeletal protein that has been Overall, our findings suggest a link among metabolic responses, shown to aggregate in cells that lack activity of small heat shock SG formation, and proteome solubility remodeling involving protein HspB7 (61). We also observed a binding partner of Flnc, about a fifth of the proteome and reveal a modular organization Desm, to be more soluble in four stresses (but, of note, was not of the protein homeostasis system that regulates metastable seen in all of the stress datasets). This result would be expected if proteins against aggregation. Flnc misfolds and aggregates and is unable to act as an appro- priate scaffold to interacting partners. Further evidence is that Methods other that cause myofibrillar myopathies when mutated Expanded details are provided in SI Appendix, Supplementary Methods. are Desm and chaperones DnajB6, HspB8, Cryab, and Bag3 which suggest these chaperones are critical to stabilizing the Stress Conditions. Neuro2a cells were cultured in a medium with the following stress treatments: 20 μM Ver155008 (Sigma-Aldrich) for 18 h; 800 μM no- folded state of Flnc (62). We also noted Bag3, which also vobiocin (Sigma-Aldrich) for 6 h; 5 μM MG132 (Sigma-Aldrich) for 18 h; colocalizes in SGs, as one of our proteins that become more 500 μM sodium arsenite (Sigma-Aldrich) for 1 h; 5 μM tunicamycin insoluble in, at least, three stresses. Thus, it remains possible that (Sigma-Aldrich) for 18 h; 10 μM 17-AAG (Sigma-Aldrich) for 24 h. For Httex1 the machinery involving Bag3 and other SGs is very competent at stress, Neuro2a cells were transiently transfected with the Httex1 fusions buffering against aggregation of misfolded proteins under stress to mCherry. (63) and that Flnc might be one of the first proteins vulnerable to aggregation under prolonged stress. Cell Fractionation by Ultracentrifugation. Neuro2a cells were harvested, pel- The signatures for the stresses were generally distinct, al- leted, and frozen −80 °C. The pellets were resuspended in buffer 1 (50 mM · though we observed a strong association with SG proteins and Tris HCl, 150 mM NaCl, 1% [vol/vol] IGEPAL CA-630, 10 units/mL DNase I, and folding stress as the major activator of quality control systems. In 1:1,000 inhibitor) and lysed by extrusion. The cell lysate was then split into two, one for total protein assessment and the other pelleted at concert, these responses appear to robustly buffer misfolded 100,000 g for 20 min at 4 °C. The resulting supernatant was the collected proteins from actually aggregating and are reminiscent of find- sample. ings from a prior study that found heat shock in yeast led to an adaptive autoregulatory assembly and disassembly of protein Protein Sample Preparation for Mass Spectrometry. Some 100 μL(∼100 μg) of complexes and minimal aggregation from denatured endogenous proteins from each sample were reduced, alkylated, and treated to a stan- proteins (10). Our results extend from this finding to show that dard quantitative proteomics workflow following trypsin digestion (details such responses are generally applicable to stress and that each in SI Appendix, Supplementary Methods). Quantitation was reductive dimethyl stress provides a unique pattern of response. labeling. One of the intriguing findings was the heterogeneity and dynamism of SG proteins. There are now, at least, 238 proteins Statistical Analysis. The details of the tests were indicated in the figure legends. Significant results were defined for P < 0.05 in the figures. Dataset that have been curated to reside in SGs (22). It is apparent that S3 shows the statistical results for tests cited in the paper. there is compositional heterogeneity in the assembly of SG (22, 64) and our data further suggest an even more diverse level of Data Availability. The mass spectrometry proteomics data have been de- heterogeneity and specificity to different forms of stress than posited in the ProteomeXchange Consortium via the PRIDE partner repository currently understood. with the dataset identifiers (accession nos. PXD014420 and PXD015573).

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