Pharmacoproteomics Pinpoints HSP70 Interaction for Correction of the Most Frequent Wilson Disease-Causing Mutant of ATP7B

Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B

Mafalda Concillia, Raffaella Petruzzellia, Silvia Parisib, Federico Catalanoa,c, Francesco Sircia,1, Francesco Napolitanoa,2, Mario Rendaa, Luis J. V. Galiettaa,d, Diego Di Bernardoa,e, and Roman S. Polishchuka,3

aTelethon Institute of Genetics and Medicine, Pozzuoli, Naples 80078, Italy; bDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy; cInstitute of Biosciences and Bioresources, Consiglio Nazionale delle Ricerche (CNR), Naples 80131, Italy; dDepartment of Translational Medical Sciences, University of Naples Federico II, Naples 80131, Italy; and eDepartment of Chemical, Materials and Industrial Production Engineering, University of Naples Federico II, Naples 80125, Italy

Edited by Jennifer Lippincott-Schwartz, Janelia Farm Research Campus, Ashburn, VA, and approved November 12, 2020 (received for review April 14, 2020) Pathogenic mutations in the copper transporter ATP7B have been emerged as an attractive strategy to rescue ATP7B mutant hypothesized to affect its protein interaction landscape contribut- phenotypes in Wilson disease (5, 8, 11, 12). ing to loss of function and, thereby, to hepatic copper toxicosis in Since the interaction landscape of mutant proteins may differ Wilson disease. Although targeting mutant interactomes was pro- from that of the wild-type (WT) protein, mutant-specific interac- posed as a therapeutic strategy, druggable interactors for rescue of tions represent potential targets for mutant correction (13–15). ATP7B mutants remain elusive. Using proteomics, we found that the For example, the interactome of cystic fibrosis transmembrane frequent H1069Q substitution promotes ATP7B interaction with conductance regulator (CFTR) with a F508del mutation, the most HSP70, thus accelerating endoplasmic reticulum (ER) degradation frequent among cystic fibrosis (CF) patients, contains quality of the mutant protein and consequent copper accumulation in he- control proteins that can be potentially targeted to treat CF (13, patic cells. This prompted us to use an HSP70 inhibitor as bait in 15). By contrast, the impact of mutations on the interactome of a bioinformatics search for structurally similar Food and Drug ATP7B remains poorly understood. Only a few interactors Administration-approved drugs. Among the hits, domperidone

(COMMD1, clusterin, and CRYAB) have been documented to be CELL BIOLOGY emerged as an effective corrector that recovered trafficking and involved in proteostatic regulation of ATP7B (11, 16, 17). How- function of ATP7B-H1069Q by impairing its exposure to the HSP70 ever, the value of these interactors for ATP7B mutant correction proteostatic network. Our findings suggest that HSP70-mediated remains questionable due to their poor druggability. Thus, the degradation can be safely targeted with domperidone to rescue demand for interactors suitable for pharmacological correction of ER-retained ATP7B mutants and, hence, to counter the onset of ATP7B mutants remains open. Wilson disease. Searching for such interactors, here we used proteomics to identify how the H1069Q mutation affects the ATP7B Wilson disease | ATP7B | mutant correction | protein quality control | copper metabolism Significance large number of inherited disorders are caused by gene Wilson disease is a disorder of copper homeostasis caused by mutations that affect folding of the corresponding protein A mutations in ATP7B. The most frequent mutation of ATP7B product and, as a consequence, lead to aberrant localization of results in an H1069Q substitution that affects the localization the mutant and/or to its rapid degradation by quality control and stability of the protein product. By interrogating the machineries (1–3). Large multispan membrane proteins repre- interactome of ATP7B-H1069Q, we found that this mutant sent a particular challenge for quality control systems as their shows stronger interaction with HSP70, which drives mutant luminal, transmembrane, and cytosolic domains require the or- degradation. Using an HSP70 inhibitor for structural similarity chestrated effort of several proteostatic mechanisms. Therefore, searches, we identified a Food and Drug Administration- these types of protein (that include numerous transporters and approved drug that increases ATP7B-H1069Q stability in cells receptors) are particularly susceptible to mutations that desta- and thus improves ATP7B function. This pharmacoproteomic bilize their native architecture and cause retention and degra- strategy provides an effective shortcut from understanding dation in the endoplasmic reticulum (ER) (2, 4). cellular mechanisms operating in Wilson disease to rapid The most frequent mutations that cause Wilson disease result identification of safe pharmacological tools and therefore in ER-retained/degraded variants of the copper-transporting might be expanded for drug repurposing to counteract other ATPase ATP7B (5, 6). ATP7B is expressed mainly in the liver genetic disorders. where it supplies copper to secreted proteins in the trans-Golgi network (TGN) or traffics toward the canalicular surface of Author contributions: R.S.P. designed research; M.C., R.P., S.P., F.C., F.S., F.N., and M.R. hepatocytes to transport excess copper into the bile (7). Al- performed research; M.C., R.P., S.P., F.C., F.S., F.N., M.R., L.J.V.G., D.D.B., and R.S.P. ana- though ER-retained mutants of ATP7B are capable of trans- lyzed data; and M.C., F.S., L.J.V.G., D.D.B., and R.S.P. wrote the paper. porting copper ions across membranes, they undergo rapid The authors declare no competing interest. degradation and fail to reach copper excretion sites (5, 6, 8). As a This article is a PNAS Direct Submission. result, copper accumulates inside hepatocytes and causes ex- Published under the PNAS license. tensive toxicity culminating in cell death, hepatitis, cirrhosis, and 1Present address: STALICLA DDS, World Trade Center, Barcelona 08039, Spain. acute liver failure (9). ER-retained variants of ATP7B comprise 2Present address: Computational Bioscience Research Center, King Abdullah University of H1069Q (∼50% in European and North American patients), Science and Technology, Thuwal 23955, Saudi Arabia. R778L (∼40% in Eastern Asian patients) and a number of less 3To whom correspondence may be addressed. Email: [email protected]. frequent isoforms, altogether constituting the largest cohort This article contains supporting information online at https://www.pnas.org/lookup/suppl/ among Wilson disease-causing ATP7B mutants (10). In this doi:10.1073/pnas.2006648117/-/DCSupplemental. context, pharmacological correction of the trafficking defect has First published December 7, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2006648117 PNAS | December 22, 2020 | vol. 117 | no. 51 | 32453–32463 Downloaded by guest on October 1, 2021 interactome. Among the mutant-specific interactors, HSP70 HSP70 Suppression Reduces ER Retention and Degradation of the emerged as a potential target for correction because HSP70 ATP7B-H1069Q Mutant. Next we investigated whether HSP70 suppression recovered the stability and trafficking of ATP7B- suppression facilitates export of ATP7B-H1069Q from the ER H1069Q. We subsequently used a specific HSP70 inhibitor to and, hence, reduces its turnover. To this end, HepG2 cells were search for structurally similar Food and Drug Administration used as a Wilson disease-relevant cell type (19), while HeLa cells (FDA)-approved drugs. One of the screen hits, domperidone, were employed because RNA interference (RNAi) works very affected the interaction of the ATP7B mutant with components efficiently in this cell type. RNAi-mediated ablation of HSP70 of the HSP70-dependent degradation machinery, facilitated ex- promoted transport of ATP7B-H1069Q to the Golgi in both port of ATP7B-H1069Q from the ER, and reduced Cu toxicity in HepG2 and HeLa cells (Fig. 2 A and B) and increased overall mutant-expressing cells. This indicates that systematic analysis of ATP7B-H1069Q levels (Fig. 2 C and D). mutant protein proteostatic networks can identify promising To test further the potential of HSP70 as a target for correc- candidates for the correction of ATP7B mutants and for drug tion of the ATP7B-H1069Q mutant, we used a highly specific repurposing in Wilson disease. allosteric HSP70 inhibitor, HS-72 (Fig. 3A). HS-72 binds C306 in the ATPase domain of two almost identical isoforms of HSP70 Results (A and B) and reduces their affinity for ATP (20). Fig. 3B shows The H1069Q Mutation Promotes ATP7B Interaction with HSP70. Al- that vitally important members of the HSP70 family (HSC70; though the ATP7B-H1069Q and WT protein interactomes have HSP70-2, -5 and -9) do not contain cysteine in the same position been investigated with traditional proteomics (8), this proteo- and hence are not sensitive to HS-72 (20). HS-72 caused sub- C mics approach does not show a difference in the binding of any stantial relocation of ATP7B-H1069Q to the Golgi (Fig. 3 and D C D given protein with either WT or mutant ATP7B (18). As a result, ) and to LAMP1-positive compartments (Fig. 3 and ) interactors that bind both ATP7B variants but exhibit preference where ATP7B normally resides upon Cu overload (19, 21). In for the mutant risk being ignored as false negatives. To cir- parallel, we tested whether HS-72 reduces ATP7B-H1069Q cumvent these drawbacks, we employed a very sensitive stable degradation. To this end, we investigated the kinetics of ATP7B- isotope labeling by amino acids in cell culture (SILAC) proteo- H1069Q degradation using cycloheximide, a protein synthesis mics approach (18) to detect the proteins that preferentially bind inhibitor. HS-72 increased the amounts of the mutant ATP7B at to the ATP7B-H1069Q mutant (Fig. 1A). HepG2 cells were both 30-min and 5-h intervals of cycloheximide (CHX) treatment F G grown in medium containing “light” (K0R0), “medium” (K4R6), (Fig. 3 and ), indicating that the HSP70 inhibitor slows down or “heavy” (K8R10) amino acids. The “medium” and “heavy” mutant degradation. cells were transduced with adenoviruses carrying complementary The HSP70 Inhibitor Facilitates Delivery of ATP7B-H1069Q to the DNA (cDNA) of either ATP7B-WT-GFP or ATP7B-H1069Q- Appropriate Cell-Surface Domain. Considering that suppression of GFP, respectively, while empty GFP was expressed in light cells HSP70 with either RNAi or the chemical inhibitor had an almost and used as a negative control in immunoprecipitation (IP) and identical impact on ATP7B-H1069Q stability/localization, we subsequent proteomics procedures (Fig. 1A). Light, medium, used HS-72 as a simpler tool to inhibit HSP70 in further ex- and heavy cell lysates were pooled and immunoprecipitated periments. First, we employed surface biotinylation (5, 19) in (Fig. 1A) with a GFP-specific antibody tested for IP in control HeLa cells to test whether HSP70 suppression increases ATP7B- experiments (SI Appendix, Fig. S1), and the individual proteins H1069Q amounts at the plasma membrane. Incubation with HS- were identified by mass spectrometry. Quantification of light 72 resulted in a significant increase in the amount of the mutant peptides from cells expressing empty GFP allowed us to protein in the cell-surface biotinylated fraction (Fig. 4 A and B), threshold all false-positive interactions. For the remaining pu- demonstrating that the inhibition of HSP70 improves delivery of tative interactors (Dataset S1), the ratio of heavy to medium B the mutant protein to the plasma membrane. peak intensities in the mass spectrum was calculated (Fig. 1 )to In hepatocytes, ATP7B has to be targeted to the canalicular reveal proteins enriched in either ATP7B-H1069Q or ATP7B- (apical) surface to support excretion of Cu into the bile (19, 22). WT interactomes (defined from hereon as “mutant-specific” or “ ” To test whether the HSP70 inhibitor facilitates delivery of WT-specific interactors). ATP7B-H1069Q to the canalicular surface, HepG2 cells were To select meaningful candidates for mutant correction, we grown to achieve maximal polarization that is manifested by the employed gene ontology (GO) analysis, which revealed strong development of the canalicular vacuole, which is formed by enrichment of the mutant interactome in quality control proteins apical membranes of two neighboring cells and recapitulates the C “ ” (Fig. 1 ). In particular, we noted the HSP70 protein binding biliary duct (23). Polarized HepG2 cells were exposed to Cu for category that suggested preferential interaction of the mutant with activation of ATP7B transport and then stained for MRP2 to C SI a subset of HSP70-associated chaperones/proteins (Fig. 1 and reveal the canalicular surface. Fig. 4C shows ATP7B-WT in the Appendix A ,Fig.S2). In this group, HSPA1A, also known as canalicular domain, while ATP7B-H1069Q failed to reach the HSP70 or inducible HSP70, looked particularly interesting for canalicular vacuole (Fig. 4D). However, exposure of these cells several reasons. HSP70 had one of the top confidence scores to the HS-72 inhibitor resulted in an increase in the amounts of Materials and Methods ( ), number of peptides, and sequence cov- ATP7B-H1069Q in the canalicular region (Fig. 4 E and F). This erage among ATP7B interactors and exhibited an almost three- suggests that HS-72 facilitates delivery of the mutant to the Cu fold increase in intensities of peptides that bind to the mutant excretion site at the apical surface of hepatic cells. compared to WT protein (Fig. 1B, arrow, and SI Appendix, Fig. S2A). Furthermore, we overlapped the lists of HSP70-related A Three-Dimensional Structure Similarity Search Reveals the FDA-Approved interactors and of ER genes promoting degradation of ATP7B- Drug Domperidone as a Corrector of Several ATP7B Mutants. Despite the H1069Q (12) and found that HSP70 was the only component that effectiveness of HS-72 in correcting the ATP7B-H1069Q, its transla- belongs to both lists (SI Appendix,Fig.S2B). This suggests that tion to clinical use might be lengthy due to the significant time/costs of HSP70 might both bind to the H1069Q mutant and drive its clinical studies. To circumvent thisproblem,weinvestigated whether degradation. Indeed, direct IP experiments revealed considerably HS-72 resembles any FDA-approved drug, which could be rapidly higher amounts of HSP70 in ATP7B-H1069Q pull-downs com- repurposed for clinical use in Wilson’s disease. We found three dif- pared to the WT protein (Fig. 1 D and F). Furthermore, higher ferent FDA-approved drugs using HS-72 as a query structure for affinity of HSP70 for the mutant was confirmed with a comple- three-dimensional (3D) ligand-based screening (24) of a collection of mentary proximity ligation assay (Fig. 1 E and G). 1,309 FDA-approved drugs (Fig. 5A). Among these hits, only

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Fig. 1. H1069Q mutation promotes ATP7B interaction with HSP70. (A) Scheme of the SILAC proteomics strategy. (B) The plot shows the outcome of the proteomics analysis with fold change (FC) of heavy to medium peak intensities for each putative interactor and corresponding significance (P value; t test). Red dots correspond to proteins with significant enrichment (>1.3 fold; P value >0.05) in the interactome of the H1069Q mutant, while green dots correspond to proteins enriched in the interactome of the WT protein. HSP70 (HSPA1A) is indicated by an arrow. (C) GO analysis revealed an enrichment in the number of quality control categories associated with protein misfolding (red bars) in the mutant interactome. (D and E) HepG2 cells expressing GFP-tagged ATP7B-WT or

ATP7B-H1069Q were treated with 200 μM CuSO4 for 2 h and processed for IP (D) or proximity ligation assay (PLA) (E). (D) Western blot shows higher levels of HSP70 in ATP7B-H1069Q pull-downs. (E) Efficiency of PLA was confirmed by the presence of the PLA signal (red spots) between ATP7B variants and the known ATP7B interactor p62 (positive control) and lack of PLA signal with TfR (negative control). ATP7B-H1069Q induced a stronger PLA signal with HSP70 thanthe WT protein. (F) The graph corresponds to the IP experiment in D and shows fold change in HSP70 bound to the H1069Q mutant compared to WT protein (n = 3 experiments; ***P < 0.001, t test). (G) Quantification of PLA experiment (E) shows a higher number of PLA dots in cells expressing the mutant protein (n = 30 cells; ***P < 0.001; t test). (Scale bar: 7 μm.).

domperidone increased ATP7B-H1069Q levels (Fig. 5B), promoted (4, 13, 15, 26, 27). A halide-sensitive yellow fluorescent protein ATP7B-H1069Q export from the ER to the Golgi (Fig. 5C), and assay (26) revealed that the established F508del-CFTR corrector facilitated delivery of the mutant to the canalicular domain in polar- VX-809 (27) induced a nearly three-fold increase in anion trans- ized HepG2 cells (SI Appendix,Fig.S3). port, while domperidone failed to rescue F508del-CFTR function We next investigated whether domperidone overcomes ER re- (SI Appendix,Fig.S4C). Finally, we tested domperidone for cor- tention of other disease-causing mutants of ATP7B and of other rection of the ΔA105-107P mutant of the manganese transporter membrane transporters such as CFTR and SLC30A10. Among SLC30A10 that causes toxic manganese overload (28), but failed to ATP7B mutants with significant catalytic activity (5, 6, 25), dom- detect any significant impact on ER retention of the SLC30A10 peridone stimulated ER export of the D765N, L776V, and, to some mutant (SI Appendix,Fig.S4D and E). extent, R778L mutants (SI Appendix,Fig.S4A and B) although a significant fraction of treated cells still contained ATP7B-R778L in Domperidone Rescues the Ability of ATP7B-H1069Q to Counteract the ER (SI Appendix,Fig.S4A and B). Then, we tested the ability Copper Toxicity. The beneficial impact of domperidone on the of domperidone to rescue the CF-causing F508del-CFTR mutant stability and localization of ATP7B mutants is expected to

Concilli et al. PNAS | December 22, 2020 | vol. 117 | no. 51 | 32455 Downloaded by guest on October 1, 2021 Fig. 2. Silencing of HSP70 decreases ER retention and degradation of ATP7B-H1069Q. (A and B) GFP-tagged ATP7B-WT or ATP7B-H1069Q were expressed in either HeLa or HepG2 cells. Cells expressing ATP7B-H1069Q were incubated with either control (siControl) or HSP70-specific (siHSP70) siRNAs and fixed and stained for TGN46. (A) In both cell types, ATP7B-WT resides mainly in the perinuclear Golgi compartment that is positive for TGN46, while the ATP7B-H1069Q mutant exhibits an ER pattern that poorly overlaps with the Golgi marker. RNAi of HSP70 reduced the ER localization of ATP7B-H1069Q and promoted delivery of the mutant to the Golgi. (B) The graph shows the impact of HSP70 silencing on colocalization (Pearson’s coefficient) of ATP7B-H1069Q with TGN46 (n = 30 cells; *P < 0.05, ANOVA). (C) Western blot reveals a higher amount of ATP7B-H1069Q in HSP70-silenced cells. (D) Quantification showing a significant increase of the ATP7B-H1069Q signal in Western blots (n = 3 experiments; *P < 0.05, t test). (Scale bar: 7 μm.)

reduce Cu toxicity. To test this, we used HepG2 lines lacking ATP7B-H1069Q with HSP70. IP experiments revealed a striking ATP7B (knockout [KO] cells) or expressing only mutant ATP7B reduction of HSP70 binding to the mutant in domperidone- variants (H1069Q cells and R778L cells). These ATP7B-deficient treated cells (Fig. 6B). cells poorly tolerate Cu (29, 30), and their death upon Cu Furthermore, we hypothesized that, being similar to the allo- overload provides a reliable readout of the loss of ATP7B steric HSP70 inhibitor, domperidone might alter the conformation function (Fig. 5D). Domperidone and HS-72 failed to improve of HSP70, thus affecting the efficacy of its cochaperones that the tolerance of KO cells to Cu but increased the survival of regulate ATP7B-H1069Q degradation. To test this hypothesis, we H1069Q cells (Fig. 5D and SI Appendix,Fig.S5). This suggests selected from the list of putative ATP7B interactors (Dataset S1) that both drugs require the ATP7B mutant protein product to all chaperones with a documented role in HSP70-mediated deg- confer resistance to Cu and, hence, rescue function of ATP7B- radation (DNAJs A1, A2, B1; HSPB1; BAG2) (4) and validated H1069Q. Dose–response experiments revealed a 1-μM concen- their binding to ATP7B-H1069Q in direct IP experiments. An tration of domperidone to be optimal for further use due to high ability to bind the mutant was clearly observed for DNAJA1 and efficacy and low toxicity (SI Appendix,Fig.S5). Notably, none of BAG2 (Fig. 6 B and C). DNAJA1 coordinates binding of mis- the tested concentrations significantly improved Cu tolerance in folded client proteins to HSP70 (4, 31). Importantly, we also R778L cells (Fig. 5D and SI Appendix,Fig.S5), likely due to the found that suppression of DNAJA1 reduced the degradation of failure of the drugs to support R778L delivery to the canalicular ATP7B-H1069Q (Fig. 6 D and E). IP experiments revealed that domain of the cells (SI Appendix,Fig.S3). This indicates the domperidone significantly increased the amount of DNAJA1 specificity of domperidone for the H1069Q mutant. bound to ATP7B-H1069Q (Fig. 6 B and C) while impairing the interaction of both ATP7B-H1069Q and DNAJA1 with HSP70 Domperidone Inhibits HSP70 Activity and Affects ATP7B-H1069Q (Fig. 6 B, C,andF). These findings suggest that domperidone Interaction with Components of the HSP70 Machinery. To investi- prevents HSP70 recruitment to the ATP7B-H1069Q/DNAJA1 gate the mechanisms that might underlie the ability of domper- complex and, hence, blocks further progression of the ATP7B idone to rescue ATP7B-H1069Q, we first investigated whether mutant through the HSP70-dependent degradation pathway (4, domperidone affects the catalytic activity of HSP70. Fig. 6A 31) (see scheme in Fig. 6G). Indeed, domperidone reduced the shows that domperidone reduced the activity of HSP70 even interaction of the ATP7B mutant with BAG2 (Fig. 6 B and C), more strongly than HS-72 at the same concentration. Then, we which acts downstream of DNAJA1 in the HSP70-mediated tested the ability of domperidone to impact the interaction of degradation cascade (4, 31) (Fig. 6G). Thus, domperidone

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Fig. 3. A specific inhibitor of HSP70 promotes trafficking of ATP7B-H1069Q and reduces its degradation. (A) Chemical structure of the specific HSP70 inhibitor HS-72. (B) Sequence alignment of HSP70 proteins in the region of HS-72 binding. Red font indicates the key position where cysteine 306 in HSP70-1A/B defines the binding site for HS-72. (C) HepG2 cells expressing either WT or H1069Q variants of ATP7B were incubated with 1 μM HS-72 for 24 h and then fixed

directly (Upper row) or after an additional 2 h treatment with 200 μM CuSO4 (+Cu; Lower row). The cells were then labeled for either TGN46 or LAMP1 as indicated. Arrows show LAMP1-positive structures, which received ATP7B in Cu-treated cells (D and E). The graphs show the impact of HS-72 on colocalization (Pearson’s coefficient) of WT or H1069Q variants of ATP7B with either TGN46 in untreated cells (D) or LAMP1 in Cu-treated cells (E)(n = 30 cells; *P < 0.05, ANOVA). (F and G) HepG2 cells expressing ATP7B-H1069Q were treated with 1 μM HS-72 for 24 h or left untreated before incubation with 100 μM CHX for either 30 min or 5 h. (F) Western blot indicates that HS-72 slowed down the decay of the ATP7B mutant protein in CHX-treated cell. (G) The graph shows a slower reduction of ATP7B-H1069Q levels in HS-72–treated cells (n = 3 experiments; *P < 0.05, t test). (Scale bar: 7 μm.)

Concilli et al. PNAS | December 22, 2020 | vol. 117 | no. 51 | 32457 Downloaded by guest on October 1, 2021 Fig. 4. The HSP70 inhibitor stimulates delivery of ATP7B-H1069Q to the cell surface. (A) HeLa cells expressing ATP7B-WT or ATP7B-H1069Q were treated with

10 μM HS-72 for 24 h and then with 200 μM CuSO4 for 2 h. The cells were subsequently processed for cell-surface biotinylation. (B) Quantification of the ATP7B signal in the biotinylated fraction reveals an increase in the amount of ATB7B-H1069Q in HS-72–treated cells (n = 3 experiments; *P < 0.05, ANOVA). (C–E)

Polarized HepG2 expressing either WT (C) or H1069Q (D and E) were exposed to CuSO4 for 4 h and stained with the canalicular marker MRP2. HS-72 (10 μM) was added to the ATP7B-H1069Q–expressing cells before exposure to Cu (E). Arrows and arrowheads in all images indicate an MRP2-positive canalicular vacuole (cyst). (F) The graphs show intensities of ATP7B (green) and MRP2 (red) signals along the lines 1, 2, and 3 drawn through the canalicular vacuole in C, D, and E, respectively. (Scale bars, C–E:4.8μm.)

32458 | www.pnas.org/cgi/doi/10.1073/pnas.2006648117 Concilli et al. Downloaded by guest on October 1, 2021 A B Domperidone (μM) Domperidone

ATP7B - HS-72 (query) GAPDH -

Score: 0.59 ATP7B7B-H1069QH1069Q C TGN46

Gavesnel

FDA-approved DB (1309 drugs)

Score: 0.54 Control 30.6㼼11.8%

ATP7B7B-H1069QH1069Q TGN46 Nialamide

Score: 0.59 Domperidone (10 μM) 60.8㼼8.8% D 120% WT 120% KO 120% H1069Q120% R778L 100% 100% 100% 100% 80% 80% * 80% 80% * 60% 60% 60% 60%

Viability 40% 40% 40% 40% 20% 20% 20% 20%

0% 0% 0% 0% CELL BIOLOGY

Fig. 5. The FDA-approved drug domperidone rescues localization and function of ATP7B-H1069Q. (A) HS-72 was used as a query structure for 3D ligand- based screening of a collection of FDA-approved drugs (Materials and Methods). Three hits were retrieved with a structural similarity score above 0.5. Overlapping of the query 3D structure poses (wireframe in gray) based on the superimposition of the hydrophobic (solid green; DRY probe) and polar interaction fields (solid cyan, OH2 probe) of the query and the three FDA-prioritized drugs (green mesh for hydrophobic and cyan mesh for polar interaction fields). (B and C) HepG2 cells expressing ATP7B-H1069Q were treated with domperidone (at the indicated concentrations) for 24 h and further processed for Western blot (B) or confocal microscopy (C). Percentage in C indicates colocalization (Pearson’s coefficient) of ATP7B-H1069Q with TGN46 (n = 30 cells; P <

0.001, t test). (D) Survival of WT, ATP7B-KO, ATP7B-H1069Q, and ATP7B-R778L HepG2 cell lines after treatment with 1 mM CuCl2 alone or in combination with either 1 μM domperidone or 1 μM HS-72 was analyzed using MTT assay. The MTT signal in the treated cells was normalized to those in control cells (treated with dimethylsulfoxide only) and expressed as percentage of viability (n = 3 experiments; *P < 0.05, ANOVA). (Scale bars: 6 μm.)

affects the ability of the HSP70 proteostatic network to trap (Fig. 7C and SI Appendix,Fig.S6A). We found that domperidone ATP7B-H1069Q in the ER, thereby increasing the chances of the and HS-72 improved colocalization of the mutant with a Golgi mutant to avoid ER degradation. marker (SI Appendix,Fig.S6B and C) and increased ATP7B- H1069Q levels in the Golgi compartment (Fig. 7C and SI Appendix, Domperidone Attenuates Degradation of ATP7B-H1069Q in Hepatocyte-like Fig. S6D), indicating that both drugs allow higher amounts of the Cells Derived from Patients with Wilson Disease. A growing body of ev- mutant to be correctly transported in patient cells. idence indicates that validation of pharmacological correctors requires reliable cell/animal systems with endogenous expression of the Discussion disease-causing mutant (21, 32). Such an animal system is not avail- The interactomes of misfolded pathogenic mutants have been able for ATP7B-H1069Q, but the generation of hepatocyte-like cells extensively explored to find targets for mutant protein rescue (HLCs) derived from homozygous patients with the H1069Q muta- (13–15). Despite the large number of mutant-specific interactors tion was reported recently (21). These HLCs endogenously express (13–15), only a few of them were further advanced to preclinical ATP7B-H1069Q, which, however, undergoes rapid and extensive studies with pharmacological antagonists or agonists. In part, this degradation in the ER. Only a small fraction (20%) of the mutant is because the remaining mutant interactome was either poorly escapes from the ER and reaches the TGN (21). Thus, we tested the druggable or lacked a specific inhibitor that could be used potential of both domperidone and HS-72 to protect the mutant from without the risk of significant side effects (4). degradation in patient’sHLCs.Fig.7A shows that the amount of the Here, we present a pharmacoproteomic strategy that identifies ATP7B protein product in patient HLCs was very low compared to an FDA-approved corrector of an ATP7B mutant starting from a HLCs from control subject expressing ATP7B-WT. Both domper- comparison of the H1069Q- and WT-ATP7B interactomes. To idone and HS-72 treatment resulted in a significant increase in find a possible target for mutant correction, we prioritized the ATP7B-H1069Q levels (Fig. 7 A and B), indicating that these drugs translational value of mutant-specific interactors. Apart from the attenuate mutant degradation in patient HLCs. Finally, we checked strength of interaction, the druggability and safety was consid- whether protection of ATP7B-H1069Q by domperidone (or by HS- ered for each specific binding partner of ATP7B-H1069Q. Using 72) supports mutant trafficking to the Golgi. In untreated cells, low these criteria, HSP70 emerged as an attractive and realistic amounts of the mutant might be detected in the Golgi and in the ER candidate for mutant correction. HSP70, being an ATPase, can

Concilli et al. PNAS | December 22, 2020 | vol. 117 | no. 51 | 32459 Downloaded by guest on October 1, 2021 Fig. 6. Impact of domperidone on the interaction of ATP7B-H1069Q with HSP70 and its cochaperones. (A) HSP70 ATPase activity assay reveals that HS-72 and domperidone (both at 1 μM concentration) significantly reduce the activity of HSP70 (n = 3 experiments; *P < 0.05, ANOVA). (B) ATP7B-H1069Q was immunoprecipitated from control and domperidone-treated HepG2-H1069Q cells and the pull downs (IPs) were immunoblotted for HSP70, DNAJA1 and BAG2. (C) Graph shows protein fold change in IP fractions (n = 3 experiments; **P < 0.01, t test). (D) DNAJA1 was silenced in HepG2-H1069Q cells with pooled siRNAs. Western blot revealed an increase in the ATP7B-H1069Q signal in DNAJA1-silenced cells. (E) Graph shows ATP7B-H1069Q fold change in DNAJA1- silenced cells (n = 3 experiments; **P < 0.01, t test). (F) HSP70 was immunoprecipitated from control and domperidone-treated HepG2-H1069Q cells and the pull-downs (IPs) were immunoblotted for DNAJA1. (G) A scheme of HSP70-mediated degradation [adapted from Young (4)] with ATP7B-H1069Q as a client protein with the following steps: 1—recognition of ATP7B-H1069Q by DNAJA1; 2—recruitment of HSP70 to the DNAJA1/ATP7B-H1069Q complex; 3— hydrolysis of ATP by HSP70; 4—release of DNAJA1 from the HSP70/ATP7B-H1069Q complex and BAG2-mediated ADP to ATP exchange on HSP70; 5—release of HSP70 and delivery of ATP7B-H1069Q for proteasomal degradation. Domperidone blocks step 2.

be easily suppressed with pharmacological tools (20). Further- rescue of the ATP7B-H1069Q mutant. Domperidone allowed more, despite being strongly involved in proteostasis, HSP70 the mutant to avoid recognition by the HSP70 proteostatic network activity does not seem to be indispensable since mice with and promoted ATP7B-H1069Q export from the ER into the secre- knockout of both HSP70 isoforms (A and B) are viable and tory pathway. As a result, tolerance of ATP7B-H1069Q–expressing fertile (33). This contrasts with other ATP7B interactors from cells to Cu was significantly improved by domperidone treatment. the HSP70 family, BIP and HCS70, whose depletion results in Domperidone is a dopamine-2 receptor antagonist with an excellent lethal phenotypes (33). safety profile and is widely used against gastric pain and vomiting However, targeting of HSP70 would be of limited use without a (34). Unfortunately, it was impossible to test this drug in vivo due to specific inhibitor. In recent years, a few inhibitors of HSP70 have the lack of an appropriate animal model expressing the mutant. In been explored for F508del-CFTR correction, but their specificity this context, hepatic cells from ATP7B-H1069Q homozygous patients was questionable as they also suppressed HSC70 (4). Recently, a represent a unique reliable system since they endogenously express specific inhibitor, HS-72, was found to target HSP70 on the C306 the ATP7B mutant and might be used for preclinical drug testing residue (20) that is not shared by other vitally essential HSP70 (21). Importantly, the effectiveness of domperidone was validated in proteins. We demonstrated that HS-72 was effective in reducing this patient-derived cell system, indicating that the drug has the po- degradation, rescuing trafficking, and correcting localization of tential to be rapidly repurposed for clinical use. ATP7B-H1069Q. Considering that the inhibitor is well tolerated Could domperidone be used to rescue other disease-causing in vivo (20), it might become a promising candidate for therapy. mutants? We found that domperidone was unable to correct ER- Unfortunately, translation from preclinical studies to the pa- locked variants of CFTR and SLC30A10 and exhibited a high tient bedside frequently takes an excessively long period for any degree of selectivity for different ATP7B mutants. It seems that new molecule (such as HS-72). In this context, we reasoned that such selectivity was not defined by location of the mutation in a already approved drugs with a chemical structure resembling particular domain of ATP7B, indicating that domperidone does HS-72 might correct ATP7B-H1069Q and thus provide a not inhibit a domain-specific ER degradation (ERAD) pathway, shortcut for transition into clinical use. Using in silico screening, such as ERAD-M or ERAD-C (35). For example, of the variants we identified the FDA-approved drug domperidone as an effi- with mutations in the fourth transmembrane domain of ATP7B, cient replacement of HS-72 for inhibition of HSP70 and for D765N and L776V were recovered from the ER by domperidone

32460 | www.pnas.org/cgi/doi/10.1073/pnas.2006648117 Concilli et al. Downloaded by guest on October 1, 2021 CELL BIOLOGY

Fig. 7. Domperidone and HS-72 rescue endogenous ATP7B-H1069Q in hepatic cells obtained from patients with Wilson disease. HLCs from patients homozygous for the H1069Q mutation in ATP7B were treated with 1 μM HS-72 or 1 μM domperidone and prepared for either Western blot (A and B)or immunofluorescence (C). HLCs from control subject expressing ATP7B-WT were used as control. (A) Western blot shows an increase in ATP7B-H1069Q levels in patient HLCs, which were treated with HS-72 or domperidone. (B) Quantification of Western blots reveals a significant increase in the ATP7B-H1069Q signal upon HS-72 and domperidone treatment (n = 3 experiments; *P < 0.05, ANOVA). (C) Control and patient HLCs were labeled for ATP7B and the TGN marker Golgin 97. Confocal images showing high levels of the ATP7B signal in the TGN of control HLCs and low ATP7B levels in the TGN of patient HLCs (arrows). Both HS-72 and domperidone increase ATP7B-H1069Q amounts in the TGN of patient HLCs. (Scale bar: 10 μm.)

treatment, while rescue of the R778L mutant was fairly poor. Indeed, our data indicate that the sets of HSP70/HCS70 Similar differences in sensitivity to domperidone were noted cochaperones operating in the degradation of ATP7B-H1069Q between the H1069Q and L1083F mutants, which both carry a differ from those that have been reported to drive degradation of substitution in the nucleotide-binding domain of ATP7B. This F508del-CFTR. We found that DNAJA1 recognizes ATP7B- suggests that recognition/degradation of different mutants relies H1069Q and directs the mutant into the HSP70-dependent on diverse and specific sets of proteostatic molecules and that degradation pathway. Indeed, domperidone inhibits the pro- domperidone targets only some of these sets. gression of the ATP7B-H1069Q/DNAJA1 complex through this

Concilli et al. PNAS | December 22, 2020 | vol. 117 | no. 51 | 32461 Downloaded by guest on October 1, 2021 pathway by blocking the interaction of both the ATP7B mutant sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The lanes were and DNAJA1 with HSP70 (Fig. 6G). By contrast, F508del- excised from the gel and sent to Oxford University Proteomics Facility, where CFTR degradation is promoted by different DNAJ proteins they were subsequently digested and subjected to nanoscale liquid chro- (B12 and C5) and requires HCS70 instead of the HSP70 (36, 37). matography coupled to tandem mass spectrometry (nano LC-MS/MS; see SI Thus, domperidone is unlikely to impair the HCS70 proteostatic Appendix, Supplementary Materials and Methods, for details). The nano LC- MS/MS data were acquired in the Orbitrap using Xcalibar v2.1 software network driving degradation of F508del-CFTR. (Thermo Fisher Scientific). The raw data files were processed and quantified On the other hand, such selectivity indicates that domperidone using Proteome Discoverer software v1.2 (Thermo Fisher Scientific). All data should not significantly affect overall protein quality control sur- were filtered to satisfy a false discovery rate of <5%. For each putative veillance. Indeed, the drug did not impact trafficking and delivery interactor the score was calculated by Proteome Discoverer (using the of both secreted and membrane proteins in hepatic cells (SI Ap- MudPIT scoring method) as a measure of confidence in the identification of pendix,Fig.S7). Therefore, the eventual risks of using domper- the protein. Several parameters such as score, coverage, and number of idone in patients are expected to be minimal and consistent with peptides were considered for interactor ranking. Most importantly, the ra- its excellent biosafety properties (34). Existing Wilson disease tios between heavy/light, medium/light, and medium/heavy peak intensities therapies (zinc salts and Cu chelators) may cause severe side ef- in the mass spectrum were calculated for each putative interactor to avoid fects and intolerance or even be ineffective in a substantial cohort false-positive hits and to reveal enrichments in either ATP7B-WT or ATP7B- of patients (9, 38). Therefore, domperidone offers an option for H1069Q interactomes. reduction of dosage of these drugs (or even their substitution) in patients expressing ATP7B-H1069Q or similar domperidone- Drug Treatments. HS-72, a specific chemical inhibitor of HSP70 (HSPA1A/B), was provided by Timothy Haystead (Duke University School of Medicine, sensitive ATP7B mutants. This does not mean that the correc- Durham, NC) (20). To investigate the impact of HS-72 and the structurally tion approach would not work in patients with other missense ATP7B similar FDA-approved drug domperidone (see chemical similarity search in SI mutations of . A significant effort should be made to an- Appendix, Supplementary Materials and Methods) on stability and locali- alyze the interactomes of these ATP7B mutants to detect specific zation of ATP7B-H1069Q, different cell types expressing the mutant were binding partners, which in turn could be targeted for mutant treated with different concentrations of the drugs (ranging from 0.5 to rescue and further identification of bio-safe correctors. 100 μM) for 24 h and then prepared for immunofluorescence or Western blot. Minimal effective concentrations of the drugs were than tested for Materials and Methods their ability 1) to rescue copper-dependent trafficking of ATP7B-H1069Q, 2) Antibodies, cDNAs, Adenoviruses, and RNAi. The full list of antibodies, cDNAs, to reduce Cu toxicity in ATP7B-H1069Q–expressing cells, and 3) to correct and small interfering RNA (siRNAs) are provided in SI Appendix, Supple- other ER retained mutants of ATP7B, CFTR, or SLC30A10. To analyze the mentary Materials and Methods. Adenoviruses carrying cDNAs of dynamics of ATP7B-H1069Q stabilization, HS-72–treated and control cells ATP7B-WT-GFP or ATP7B-H1069Q-GFP (8, 19) were used at different values were incubated with 100 μg/mL cycloheximide for different time intervals of multiplicity of infection to transduce HepG2 or HeLa cells. For RNAi, and then were processed for Western blot. HepG2 or HeLa cells were transfected with individual or pooled siRNAs using Dharmafect4 (Dharmacon) or Oligofectamine (Invitrogen) according to the Other Methods. Gene ontology enrichment analysis, chemical similarity manufacturer’s instructions. Silencing efficiency was evaluated using search, Western blot, immunoprecipitation, cell-surface biotinylation, prox- Western blot. imity ligation assay, immunofluorescence, confocal microscopy as well as assays for Cu-dependent trafficking of ATP7B, cell viability, and activities of Cell Culture. HeLa and HepG2 cells were grown in HepG2 cells were grown in HSP70 and CFTR are described in detail in SI Appendix, Supplementary Dulbecco’s Modified Eagle Media (DMEM). ATP7B-deficient HepG2 cells (KO, Materials and Methods. H1069Q, and R778L) were provided by Hartmut Schmidt (Medizinische Klinik B für Gastroenterologie und Hepatologie, Universitätsklinikum Münster, Statistical Analyses. Data are expressed as mean ± SD, collected from multiple Münster, Germany) and grown in RPMI medium 1640. HLCs from patients independent experiments performed on different days. Statistical signifi- carrying the H1069Q mutation and from control subjects were differenti- cances for all data were computed using Student t tests or one-way ANOVA ated from corresponding induced pluripotent stem cell clones according to (for all figures, *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistical published protocols (21). The bronchial epithelial cell line CFBE41o- significance). expressing F508del-CFTR and halide-sensitive yellow fluorescent protein (HS-YFP) (26) was kept in Minimum Essential Media (MEM). All cell lines were Data Availability. The mass spectrometry proteomics data have been de- supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin, and posited in the ProteomeXchange Consortium via the PRIDE partner repository streptomycin. with the dataset identifier PXD016816 (39). SILAC Proteomics. HepG2 cells were grown in DMEM containing medium ACKNOWLEDGMENTS. We thank Cathal Wilson for critical reading of the R6K4, heavy R10K8, or light R0K0 isotopes (Dundee Cell Products). Medium, manuscript and acknowledge support from Telethon Institute of Genetics heavy, and light cells were then transduced with ATP7B-WT-GFP, ATP7B- and Medicine bioinformatics and microscopy cores. This work was funded by H1069Q-GFP, or GFP (control) adenoviruses, respectively, and incubated with Telethon, Italy, Grant TIGEM-CBDM9 (to R.S.P.); Consiglio Nazionale delle lysis buffer. The GFP signal was analyzed with Western blot in lysates from Ricerche/Russian Foundation for Basic Research Collaboration Program, Italy- HepG2 cells expressing ATP7B-H1069Q-GFP, ATP7B-WT-GFP, or GFP alone. Russia Grant 18-515-7811; PRIN-2017 (2017CH4RNP) (to S.P.); the Wilson The samples were then pooled in a 1:1:1 ratio according to the GFP quan- Disease Association; Associazione Nazionale Malattia di Wilson; and a tification, immunoprecipitated with anti-GFP antibody, and processed with Veronesi Foundation Fellowship (to R.P.).

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