Parasite proteostasis and artemisinin resistance

The continued emergence and spread of resistance to artemisinins, the cornerstone of first line antimalarials, threatens significant gains made toward malaria elimination. Mutations in Kelch13 have been proposed to mediate artemisinin resistance by either reducing artemisinin activation via reduced parasite hemoglobin digestion or by enhancing the parasite stress response. Here, we explored the involvement of the parasite unfolded protein response (UPR) and ubiquitin proteasome system (UPS), vital to maintaining parasite proteostasis, in the context of artemisinin resistance. Our data show that perturbing parasite proteostasis kills parasites, early parasite UPR signaling dictate DHA survival outcomes, and DHA susceptibility correlates with impairment of proteasome-mediated protein degradation. These data provide compelling evidence toward targeting the UPR and UPS to overcome existing artemisinin resistance.

The exact role of Kelch13 in artemisinin resistance is an ongoing area of study. Two non-mutually exclusive hypotheses have been put forward to explain Kelch13-mediated artemisinin resistance: (1) decreased artemisinin activation via reduced hemoglobin digestion, and (2) enhanced stress response to counter artemisinin-mediated protein and lipid damage. Knock sideways studies point to a role for Kelch13 in endocytosis of hemoglobin, as parasites in which > 60% 32 of Kelch13 is mislocalized display both reduced uptake of uorescent dextran 33 and lower abundance of hemoglobin-derived peptides 34 .
Omics studies point to a role for Kelch13 in the parasite stress response. Transcriptomics of artemisinin resistant clinical isolates were found to upregulate genes involved in protein folding, protein repair, and proteasome subunits 35 . In addition, the lab-adapted clinical isolate Cam3.II Kelch13 R539T and its isogenic counterparts Cam3.II Kelch13 WT and Cam3.II Kelch13 C580Y were examined by transcriptomics and proteomics, revealing that artemisinin-resistant (Kelch13 mutant) parasites express higher levels of genes involved in the ubiquitin proteasome system (UPS), redox, and intracellular vesicles (DHA), the active metabolite of all clinical artemisinins, and the related peroxide OZ439 (also known as artefenomel) 55 . This increase in sensitivity was not only observed at the early ring stage where artemisinin resistance is classically observed, but also throughout the asexual life cycle 55 . These data suggest that the proteasome is critical for parasites to survive artemisinins and acts in a manner distinct from Kelch13. We and others have shown that proteasome inhibitors synergize with DHA to potently kill artemisinin-resistant P. falciparum in vitro and in vivo 56, 57 . Aside from DHA, proteasome inhibitors also synergized with distinct antimalarial compounds such as the peroxide OZ439, the deubiquitinase inhibitor b-AP15, and the redox inhibitor methylene blue, which are structurally diverse and possess distinct antimalarial modes of action 57 . Given the crucial role of proteasomes in restoring proteostasis, we were intrigued if the observed synergy was due to additional perturbation of proteostasis mechanisms by these synergistic compounds. To interrogate the role of proteostasis mechanisms in parasite artemisinin response and resistance, we examined UPR kinetics and proteasome activity in Kelch13 mutants and proteasome mutants. Our data show that Kelch13 WT and Kelch13 mutant parasites display distinct stage-dependent UPR kinetics. Importantly, early responses of hyperactivation and a concomitant unresolved UPR dictate eventual death in artemisinin-sensitive Kelch13 WT parasites.
Finally, we show that a well-functioning proteasome promotes parasite survival to artemisinin, independent of the canonical K13-mediated resistance pathway.

Antimalarial compounds synergistic with proteasome inhibitors disrupt proteostasis
We previously showed that the P. falciparum-speci c proteasome inhibitors WLL and WLW synergize with four of sixteen candidate and clinically used antimalarials 58 . The four synergistic compounds (DHA, OZ439, b-AP15, and methylene blue) are structurally diverse and have distinct modes of action. DHA and OZ439 non-speci cally alkylate nearby proteins 5,6 , b-AP15 inhibits a proteasome-associated deubiquitinase 59 , and methylene blue interferes with redox homeostasis 60,61 . We were curious why these different classes of antimalarials were synergistic with proteasome inhibitors, and hypothesized that they may perturb proteostasis. To this end, Cam3.II Kelch13 WT parasites were synchronized to 26-30 hpi trophozoite stages and treated for 6 h with a 5x IC 50 concentration of the proteasome inhibitor WLL 56 , the synergistic compounds DHA, OZ439, b-AP15, and methylene blue, the antagonistic compound chloroquine, or the vehicle control, DMSO. UPR activation was determined by levels of p-eIF2α, a marker of UPR activation, normalized to total eIF2α levels 62 . Proteasome dysfunction was determined by levels of K48-linked ubiquitination 63 normalized to BiP, because in Cam3.II strain parasites BiP does not increase in response to DHA 36 . Treatment with the synergistic compounds DHA, OZ439, and b-AP15 all resulted in UPR activation with OZ439 resulting in the greatest UPR activation followed by DHA and b-AP15 treatment yielding similar levels of UPR activation (Fig. 1a, b, and Supplementary Fig. 1). These three compounds led to an accumulation of K48-linked ubiquitination, and the effect on ubiquitination from each of these compounds was similar (Fig. 1a, c, and Supplementary Fig. 1). Methylene blue, which was synergistic with proteasome inhibitors in ring stages but additive in trophozoite stages 58 , did not activate the UPR but led to a 2-fold increase in K48-linked ubiquitination, although this was not statistically signi cant (p = 0.2014; Fig. 1a-c and Supplementary Fig. 1). In contrast, the antagonistic compound chloroquine 58 , which inhibits heme detoxi cation 64 , did not alter levels of p-eIF2α or K48linked ubiquitination relative to the DMSO-treated control (Fig. 1a-c and Supplementary Fig. 1). As a positive control for proteasome inhibition, parasites were treated with WLL. Indeed, WLL-treated parasites accumulated high levels of K48-linked ubiquitination (Fig. 1a, c, and Supplementary Fig. 1). A more moderate UPR activation was observed with WLL treatment, corroborating the primary effect on proteasome inhibition leading to the secondary effect of UPR activation 65,66 . Together, these data indicate that compounds that synergize with proteasome inhibitors to potently kill malaria parasites disrupt proteostasis, and suggest that the proteasome is important for parasite proteostasis restoration.
Kelch13 WT and Kelch13 mutant parasites differentially regulate the UPR The UPR is an exquisitely well-regulated process, and we were interested in understanding the kinetics of UPR activation and resolution in artemisinin-sensitive and artemisinin-resistant parasites. To do so, Cam3.II Kelch13 WT (hereon referred to as WT; Table 1) and Cam3.II Kelch13 R539T parasites (hereon referred to as R539T; Table 1) were tightly synchronized to 0-3 hpi rings and treated with the physiologically-relevant concentration of 700 nM DHA for 3 h, mimicking conditions of the RSA used to delineate artemisinin resistance in vitro 28 (Fig. 2a, top and middle panels). In response to DHA, levels of p-eIF2α increased 1.5-fold in both parasites. However, only DHA-treated WT parasites had signi cantly higher levels of p-eIF2α compared to mock-treated controls and relative to the DHA-treated R539T mutant ( Fig. 2b, c, Supplementary Fig. 2a).
Next, UPR resolution was monitored in these parasites following drug removal. Levels of p-eIF2α declined over time in both parasites following DHA washout (Fig. 2d, e, f, Supplementary Fig. 2b). However, by 6 h post washout, levels of p-eIF2α in WT parasites remained elevated relative to the mock-treated control ( Fig. 2d and e), suggesting that these parasites were unable to resolve the UPR and remained in a state of stress. In contrast, at 6 h post-washout, levels of p-eIF2α in R539T parasites returned to basal levels ( Fig.  2d and Fig. 2c-e). Interestingly, C580Y parasites with an additional β2 C31Y mutation, which sensitized parasites to DHA 68 , also had elevated levels of p-eIF2α at 6 h post-washout compared to mock-treated counterparts ( Supplementary Fig. 2c-e). Together, the data suggest that parasites sensitized to DHA are unable to resolve DHA-mediated UPR activation despite removal of the drug.
To determine if these phenotypes would be maintained at the trophozoite stage, UPR activation was monitored in WT and R539T parasites synchronized to 26-30 hpi trophozoites ( Fig. 2A, bottom panel). Upon treatment with 50 nM DHA, a 5x IC 50 concentration, the UPR was activated in both parasites in a time-dependent manner. Of note, levels of p-eIF2α were signi cantly higher at 3 h in R539T vs. WT parasites (Fig. 2g, h, and Supplementary Fig. 2f), suggesting a more robust UPR activation in the R539T mutant. By 6 h post-treatment, levels of p-eIF2α were similar between the parasites examined ( Fig. 2g, h, and Supplementary Fig. 2f). These data show that the kinetics of UPR activation and resolution are dependent on both Kelch13 genotype and the parasite stage during its intraerythrocytic development cycle.
Peroxides DHA and OZ439 inhibit parasite proteasome activity Previously it was shown that DHA inhibits β5 proteasome catalytic activity in artemisinin-sensitive Kelch13 WT parasites and leads to an accumulation of ubiquitinated proteins 8,69 . Since artemisininresistant and Kelch13 mutant parasites have been shown to express higher levels of proteasome subunits 35,36 , we sought to determine whether Kelch13 mutations impacted DHA-mediated proteasome inhibition. Although the β5 catalytic activity is responsible for the majority of protein degradation, we were also interested in the effect of DHA on the other two catalytic subunits of the proteasome as they play a role in protein degradation, in addition to the effect of the related peroxide OZ439 on proteasome catalytic activity. Proteasome activity in DHA-treated WT, R539T, and C580Y parasites was examined using two orthogonal approaches. For both approaches, trophozoite stages were assayed since the UPS is upregulated at the trophozoite stage 46,70 and artemisinin treatment does not produce a detectable increase in ubiquitination at the early ring stage 69 . In the rst approach, proteasome subunit catalytic activity was examined in DHA-treated and OZ439-treated trophozoites using the uorogenic substrates Ac-nLPnLD-AMC, Ac-RLR-AMC, or Suc-LLVY-AMC to assess caspase-like, trypsin-like, and chymotrypsinlike activity, respectively 71 . Though these uorogenic substrates can react with other proteases in the parasites, for simplicity we will refer to caspase-like activity as β1 activity, trypsin-like activity as β2 activity, and chymotrypsin-like activity as β5 activity. WLL, a P. falciparum-selective proteasome inhibitor with activity against β2 and β5 active sites 56 was used as a positive control for inhibiting these two catalytic sites. No known inhibitor of plasmodial β1 exists, though high concentrations of WLL have been shown to moderately inhibit plasmodial β1 activity 56 .
DHA inhibited β1 (Fig. 3a), β2 (Fig. 3b), and β5 (Fig. 3c) activity in WT, R539T, and C580Y trophozoites in a statistically signi cant and concentration-dependent manner. β1 and β2 activity were inhibited by approximately 30% and 40% following treatment with 50 nM DHA and 700 nM DHA, respectively ( Fig. 3a and b). β5 activity was inhibited to the greatest extent, with approximately 40% and 60% inhibition upon treatment with 50 nM DHA and 700 nM DHA, respectively (Fig. 3c). In addition to comparing treated to untreated counterparts as detailed above, we also tested for differences in DHA-mediated inhibition depending on Kelch13 genotype but no signi cant difference in catalytic inhibition was detected between Kelch13 WT and Kelch13 mutant parasites. The DHA-related peroxide OZ439 did not inhibit β1 activity of proteasomes isolated from all tested parasite strains (Fig. 3d). Intriguingly, OZ439 modestly inhibited β2 activity (10-15% inhibition) of proteasomes derived from R539T and C580Y but not WT parasites (Fig.  3e). In addition, OZ439 selectively inhibited β5 activity (approximately 25% inhibition) of proteasomes derived from Kelch13 mutants, which was determined to be statistically signi cant at the peak plasma concentration of 3 µM OZ439 72,73 (Fig. 3f).
Although uorogenic substrate assays accurately determine proteasome catalytic activity, these assays are unable to measure proteasome-mediated protein degradation. Thus, in a second approach to measure proteasome activity, we examined the accumulation of K48-linked ubiquitination, which is a hallmark of proteasome dysfunction. Synchronized WT, R539T, and C580Y strain parasites at the 26-30 hpi trophozoite stages were treated with 50 nM DHA for up to 6 h, then lysates were examined for protein ubiquitination. In response to DHA, all parasites showed a statistically signi cant accumulation of K48linked ubiquitination in a time-dependent manner (Fig. 3g, h, Supplementary Fig. 3a). At each timepoint, levels of ubiquitination was similar across all parasites tested regardless of Kelch13 genotype ( Supplementary Fig. 3b), re ecting results obtained from proteasome catalytic activity assays. Collectively, these data show that DHA equally inhibits proteasomes from WT, R539T, and C580Y. In contrast, OZ439 selectively inhibits the β5 catalytic activity of proteasomes derived from R539T and C580Y parasites.
Mutations in 19S proteasome subunits increase parasite susceptibility to DHA Previously, we reported that an additional mutation in the 20S β2 proteasome subunit at either C31Y or C31F in the context of a C580Y background increased parasite susceptibility to DHA 55 . These parasites were generated via in vitro selection studies with the P. falciparum-speci c proteasome inhibitor WLW 58 . In the same WLW selection study, three 19S proteasome mutants were also selected for in the Cam3.II background: Cam3.II Kelch13 WT Rpt4 E380*, Cam3.II Kelch13 WT Rpn6 E266K, and Cam3.II Kelch13 C580Y Rpt5 G319S 58 (hereon referred to as Rpt4, Rpn6, and Rpt5, respectively; Table 1). Rpt4 and Rpt5 are ATPase subunits in the 19S RP base, which mediate gate opening to allow substrates into the 20S 49 .
Rpt4 is also in contact with the 19S lid 74 . Rpn6 acts as a scaffolding protein that stabilizes the interaction between the 19S and 20S 75 (Fig. 4a). The 19S RP is important for regulating protein processing prior to proteolytic degradation within the 20S CP chamber 49 . Thus, we were interested in determining if mutations in 19S subunits in the context of a C580Y background would compromise parasite resistance to DHA and if such mutations evolved on a WT background would further hypersensitize parasites to DHA.
To this end, dose response assays were performed using early ring (0-3 hpi), trophozoite (26-30 hpi), and asynchronous cultures ( Supplementary Fig. 4).  (Fig. 4f). These data demonstrate that indeed, mutations in 19S subunits increase parasite susceptibility to DHA and can even hypersensitize parasites to DHA when 19S mutations occur on a Kelch13 WT background.
Parasites with increased peroxide susceptibility have impaired proteasome-mediated protein degradation Given the increased peroxide susceptibility of parasites harboring 19S or 20S mutations, we hypothesized that the proteasome is essential for parasite survival in the face of artemisinin and similar compounds, and that the observed sensitivity of proteasome mutants to peroxides is due to a dysfunction in proteasome-mediated protein degradation. To test this hypothesis, we examined the proteasome catalytic activity of DHA-and OZ439-treated 26-30 hpi trophozoites derived from the C580Y parental strain and cognate 20S CP mutants: Cam3.II Kelch13 C580Y β2 C31Y, Cam3.II Kelch13 C580Y β2 C31F, and Cam3.II Kelch13 C580Y β5 A20S (hereon referred to as β2 C31Y, β2 C31F, and β5 A20S, respectively; Table 1). Treatment with DHA signi cantly inhibited β1 (Fig. 5a), β2 (Fig. 5b), and β5 ( Fig. 5c) activity in all parasites tested in a concentration-dependent manner. In addition, relative to its parent, the β2 C31Y mutant displayed greater inhibition of β1 activity upon treatment with 50 nM DHA (C580Y = 20% inhibition; β2 C31Y = 49% inhibition), but no other signi cant difference was observed between DHAtreated parent and proteasome mutant parasites. OZ439 did not inhibit β1 activity (Fig. 5d), but did inhibit β2 and β5 activities in all parasites tested (Fig. 5e, f). Note that at 3 µM OZ439, the β5 catalytic site of the β2 C31F mutant was signi cantly more inhibited compared to that of the parental C580Y parasite (C580Y = 28% inhibition; β2 C31F = 52% inhibition; Fig. 5f). No other signi cant difference in catalytic inhibition was observed between parental and 20S proteasome mutants treated with OZ439.
Next, inhibition of proteasome-mediated protein degradation was evaluated in 20S mutant (Fig. 5g, h) and 19S mutant parasites (Fig. 5i-l) by assessing accumulation of K48-linked ubiquitination. DHA-treated C580Y and β2 mutants led to signi cantly increased K48-linked ubiquitination compared to mock-treated parasites (Fig. 5g, h, and Supplementary Fig. 5a). In addition, relative to their parent C580Y, both β2 C31Y and β2 C31F mutants had 1.5-to 2-fold higher levels of K48-linked ubiquitination in response to 3 h treatment of DHA (Fig. 5g, h, and Supplementary Fig. 5a). The β5 A20S mutant, which did not display altered sensitivity to DHA or OZ439 55 , had minor and statistically insigni cant increases in ubiquitination ( Fig. 5g, h, and Supplementary Fig. 5a). Derived on a genetic background expressing Kelch13 C580Y, Rpt5 mutants displayed a statistically signi cant 2-fold increase in K48 ubiquitination compared to the parental strain at basal levels without any drug treatment and at 3h DHA treatment (Fig. 5i, j, and Supplementary Fig. 5b). For the 19S mutants that were derived on a Kelch13 WT background, Rpt4 and Rpn6 mutants also showed a statistically signi cant 2-fold increase in ubiquitination compared to WT at basal levels and upon drug treatment (Fig. 5k, l, and Supplementary Fig. 5c). In addition, DHA-treated Rpt4 and Rpn6 mutants accumulated signi cantly more ubiquitination compared to mock-treated counterparts (Fig. 5k, l, and Supplementary Fig. 5c). Since we observed that the UPR was differentially activated in Kelch13 WT vs. Kelch13 mutant parasites, we were also interested in determining UPR activation kinetics in proteasome mutants. However, no signi cant difference in UPR activation was observed between parental and proteasome mutant parasites at the early ring stage ( Supplementary Fig.   2c-e) or trophozoite stage ( Supplementary Fig. S6). We note that in trophozoite stages, C580Y parental strain as well as β2 C31Y, β2 C31F, β5 A20S, and Rpt5 proteasome mutants signi cantly induced UPR activation compared to untreated counterparts, albeit the level of UPR activation was similar for parent and proteasome mutants ( Supplementary Fig. S6). Collectively, these data indicate that a defect in proteasome-mediated protein degradation underlies the heightened sensitivity of proteasome mutants to peroxides, and that this defect is not mediated by increased inhibition of proteasome catalytic subunits.
The β2 C31Y proteasome mutant is sensitized to proteasome-related inhibitors Given our observation that β2 mutants exhibited proteasome dysfunction compared to the parental C580Y as well as the β5 A20S mutant, we reasoned that in addition to DHA and OZ439, β2 mutants should also selectively display increased susceptibility to compounds that inhibit proteasome-mediated protein degradation. To test this hypothesis, C580Y, β2 C31Y, and β5 A20S strain parasites were subjected to dose response assays with epoxomicin, a non-parasite selective inhibitor of the proteasome 76 , and b-AP15, an inhibitor of a proteasome-associated deubiquitinase 59 . As negative controls, we included chloroquine and methylene blue, both of whose mechanisms of action are unrelated to the proteasome 60,61,64,77 . The C580Y parent and the β5 A20S mutant, which have similar sensitivity pro les to DHA and OZ439 55 , displayed almost identical dose-response curves in response to epoxomicin (Fig. 6a), b-AP15 (Fig. 6b), chloroquine (Fig. 6c), and methylene blue (Fig. 6d). Accordingly, C580Y and β5 A20S also had similar IC 50  Dose response curves of slow growing parasites may be shifted left due to a defect in parasite tness that is unrelated to parasite susceptibility to a particular compound. Given that the β2 C31Y mutant and its parent had similar dose response curves for chloroquine and methylene blue, it is unlikely that the increased susceptibility of this parasite to DHA, OZ439, epoxomicin, and b-AP15 is due to a tness cost. However, to rule out this possibility, parasite tness competition assays were conducted with C580Y, β2 C31Y, β2 C31F, and β5 A20S parasites. No signi cant difference was found between C580Y and the 20S proteasome mutants examined (Supplementary Fig. 7). Thus, these data demonstrate that the β2 C31Y mutant is selectively sensitive to compounds that target the proteasome.

Discussion
As the prevalence of artemisinin resistance continues to rise, it becomes increasingly urgent to delineate a mechanism of resistance to inform future drug discovery and implementation of antimalarial combination therapies. In addition to the widespread artemisinin resistance in the Southeast Asian region, recent reports of Kelch13-mediated artemisinin resistance in Rwanda and Uganda detected in the past three years is of particular concern 14,19,25 26,27,78 . We have previously shown that proteasome inhibitors effectively kill artemisinin-resistant parasites and strongly synergize with DHA 57,79 . In addition, parasites moderately resistant to proteasome inhibitors are sensitized to DHA 68 . Importantly, proteasome inhibitors are effective against Ugandan parasite isolates 80 .
The proteasome is intimately involved in the UPR and protein degradation, two pillars of proteostasis. In a well-functioning cell, UPR activation will lead to upregulation of proteasome-mediated protein degradation, and inhibition of proteasome-mediated protein degradation will lead to UPR activation 65,66 . Exploration of the kinetics of UPR activation and resolution as well as proteasome activity in isogenic parasites only differing in the loci of Kelch13 or proteasome subunits yielded some surprising results.
Firstly, our data con rmed our hypothesis that antimalarials that synergize with proteasome inhibitors such as DHA, OZ439, and b-AP15 perturb proteostasis by upregulating the UPR and inhibiting proteasome-mediated protein degradation. In contrast, antimalarials that antagonize with proteasome inhibitors such as chloroquine had no effect on these measurements of proteostasis perturbations. Interestingly, methylene blue was additive with proteasome inhibitors, and had an intermediate increase in UPR activation and ubiquitination. These data suggest that directly interfering with proteostasis mechanisms is a promising antimalarial therapeutic strategy.
Secondly, we found that early parasite responses to DHA dictate eventual survival outcomes. Transcriptomics and proteomics data point to a role for Kelch13 mutants in broadly enhancing the parasite's stress response 35,36 . However, the molecular stress response pathways involved, and a wellde ned mechanism of resistance have not been elucidated. Here we show that artemisinin-sensitive Kelch13 WT parasites hyperactivate the UPR at early ring stages, indicating that these parasites are either (1) experiencing increased levels of stress and/or (2) the UPR is dysfunctionally regulated. Mislocalization studies suggest Kelch13 mutant parasites have reduced hemoglobin uptake and digestion 33,34 , and it is hypothesized that as a consequence these parasites have reduced artemisinin activation. However, the role of Kelch13 in hemoglobin uptake appears to be restricted to the ring stage 33 . Accordingly, it would be expected that the misfolded protein load in Kelch13 mutants would be lower and less prone to trigger the UPR at the ring stage. This hypothesis is consistent with our observations that early ring stage R539T parasites display little UPR activation in response to DHA and are also able to completely resolve the UPR following drug removal. In contrast, DHA-treated WT early rings display robust UPR activation and are unable to completely resolve the UPR, as seen by residual eIF2α phosphorylation 6 h after DHA removal. This is consistent with ndings that following a 3 h pulse with 700 nM DHA on ring-stage parasites, R539T parasites begin to resume protein turnover as early as 9 h after drug removal while WT parasites do not, and the differences become more pronounced at 15 h post-drug withdrawal 34 .
However, at the trophozoite stage, where hemoglobin digestion is increased 81 and Kelch13 is not involved in hemoglobin uptake 33 , we found that the UPR was activated earlier in DHA-treated R539T parasites. Activating the UPR more quickly while in the trophozoite stages could be advantageous to Kelch13 mutants, giving them a jumpstart on mitigating protein damage, given that metabolic processes and protein abundance is greatly increased during these stages compared to ring stages 34 . However, a direct molecular link between Kelch13 and the UPR remains to be identi ed and are addressed in ongoing studies.
Previous studies demonstrate con icting data regarding UPR activation in the early ring stages of Kelch13 WT vs. Kelch13 mutants. Consistent with what we observed, Dd2 Kelch13 WT 0-3 hpi rings treated with 700 nM DHA for 15 min displayed more robust UPR activation than Dd2 Kelch13 C580Y 0-3 hpi rings 41 . However, the authors observed that Kelch13 mutants displayed elevated basal UPR activation, which is in contrast to our observation 41 . This disparity could be attributed to differences in the genetic backgrounds of the parasites examined. Of note, Dd2 was adapted to the laboratory in the 1970s prior to widespread artemisinin usage, while Cam3.II was adapted in 2010s and originated from an artemisinin-resistant isolate. In a separate study, it was observed that relative to Cam3.II Kelch13 WT 0-8 hpi rings, Cam3.II Kelch13 R539T parasites had elevated UPR activation under basal conditions and in response to a 3 h treatment with 700 nM DHA 34 . It is possible that differences between early-and midring stages could explain discrepancies between these data, and the less tightly synchronized rings in 34 could be behaving more similarly to the trophozoite stage parasites in our study.
Isogenic Kelch13 mutant vs. Kelch13 WT parasites 36 and artemisinin-resistant clinical isolates 35 have been shown to have increased levels of proteasome subunits by transcriptomics and proteomics. However, we observed no noticeable difference in proteasome activity between isogenic Kelch13 WT vs. Kelch13 mutants at basal levels or when DHA-treated when we assessed model substrate cleavage as well as cellular protein degradation. Since the proteasome is a multi-subunit complex with particular stoichiometry and assembly of subunits, upregulation of some proteasome subunits may be insu cient to modulate proteasome activity. It is also possible that the assays used here are unable to detect slight differences in proteasome activity which may be biologically relevant. Collectively, these data suggest that Kelch13 does not mediate artemisinin resistance by modulating proteasome activity but rather by modulating UPR activation and resolution. It was recently reported that Kelch13 mutant parasites undergo higher levels of autophagy than Kelch13 WT parasites under basal conditions 82 , which would aid in disposing of damaged proteins thus complementing any de ciencies in proteasome-mediated protein degradation.
Yet, the proteasome may play a critical role in non-Kelch13-mediated artemisinin response. The third major nding of our study is that parasite susceptibility to DHA, mediated by mutations in the proteasome, correlated with a dysfunction in proteasome-mediated protein degradation. Previous studies showed that upon artemisinin treatment, the artemisinin-sensitive parasites 3D7 and PL2 (artemisinin-sensitive; Kelch13 WT) had a 2-fold increase in ubiquitination while the artemisinin-resistant PL7 strain (artemisinin-resistant; Kelch13 mutant) only accumulated ~1.2-fold increased ubiquitination 69 . Note that none of these three strains are isogenic, and there are multiple genetic differences between 3D7, PL2, and PL7, including at known drug resistance modulators such as P. falciparum multidrug resistance protein 1 (PfMDR1), P. falciparum multidrug resistance protein 2 (PfMDR2), and P. falciparum chloroquine resistance transporter (PfCRT) 69 . In our study, we corroborate these earlier data and show that parasites susceptible to DHA and isogenic except for mutations in proteasome subunits display increased ubiquitination. Not all proteasome mutations and resultant proteasome dysfunction affect DHA susceptibility equally across asexual blood stages. For example, while all 19S and β2 mutants tested displayed a defect in proteasome-mediated protein degradation, 19S mutants only displayed increased susceptibility to DHA in synchronized cultures, whereas the β2 proteasome mutants displayed increased sensitivity at ring, trophozoite, and asynchronous stages 55 . These data could indicate that the 20S plays an outsized role in parasite artemisinin response. Perhaps in addition to the 20S-19S complex, the 20S-PA28 complex contributes to resolving artemisinin-mediated protein damage. This is supported by previous ndings that 3D7 parasites in which PA28 is knocked out display a 2-fold lower DHA IC 50 values at the early ring stage 54 .
Although Rpt5, Rpt4, Rpn6, and β2 proteasome mutants showed increased ubiquitinated polypeptides in response to DHA compared to parental strains, these differences were not detected when we assayed for proteasome catalytic activity as measured by cleavage of uorogenic peptidyl model substrates. One reason for this discrepancy is that the uorogenic substrates can freely diffuse into the 20S CP without processing by the 19S RP, whereas detection of K48-linked ubiquitinated proteins assesses the ability of the 26S proteasome as a whole to process and degrade proteins. Interestingly, peptidyl substrate cleavage showed that at peak plasma concentrations, OZ439 signi cantly inhibits the β5 activity of R539T and C580Y proteasomes but does not inhibit WT proteasomes. This could explain why these artemisinin-resistant parasite strains do not exhibit cross-resistance to OZ439 83,84 . OZ439 also inhibited the β5 catalytic activity of β2 C31F signi cantly more than in the parental C580Y strain. These results are in concordance with our previous data showing that β2 C31F showed the greatest decrease in RSA values in response to OZ439 68 .
It remains unknown to what degree the proteasome mutations tested here affect proteasome activity physiologically. Based on the cryo-EM structure of the P. falciparum 20S proteasome, β2 C31Y and β2 C31F were mapped near the S1 binding pocket of the β2 active site and were predicted to impair WLW binding via steric hinderance 58 . The Rpt4 E380* and Rpn6 E266K mutations fall outside of conserved domains, while G319S is located within the AAA domain of Rpt5 (Supplementary Fig. 8). This could indicate the Rpt5 mutation is more detrimental to proteasome activity than Rpt4 and Rpn6 mutations.
Consistent with this hypothesis, the Rpt5 mutant displays increased sensitivity to DHA at ring and trophozoite stages in comparison to the Rpt4 and Rpn6 mutants, which were only sensitized at the ring stage. However, without the generation of transgenic parasites, the degree of DHA sensitization conferred by particular proteasome mutations and the in uence of Kelch13 cannot be determined.
In summary, the data presented here indicate that (1) antimalarial compounds that synergize with proteasome inhibitors perturb parasite proteostasis, (2) early parasite UPR signaling in response to DHA dictate eventual survival outcomes, and (3) parasite susceptibility to DHA correlates with a dysfunction in proteasome-mediated protein degradation. We show here and previously that chemical inhibition of the proteasome and mutations in the proteasome increase parasite susceptibility to DHA regardless of Kelch13 genotype 57,68 , highlighting the crucial role of the proteasome in parasite survival to artemisinin.
These data point to the UPR and UPS, two pillars of proteostasis, as pathways that can be targeted to overcome existing artemisinin resistance.  Parasites were grown at 37°C in a Heracell™ VIOS 160i CO 2 Incubator (Thermo Fisher Scienti c) at 5% O 2 , 5% CO 2 , and 90% N 2 (Matheson Gas, Irving, Texas).

Stage synchronization
For dose response assays, early ring stages (0-3 hpi) were obtained as previously described 55 . Brie y, cultures were exposed to 5% sorbitol (Acros Organics) at 37°C for 10 min and then cultured for 33 h. Then, cultures were incubated with RPMI 1640 supplemented with 14.3 U/mL sodium heparin (Merck, Kenilworth, NJ) at 37°C for 30 min with intermittent vortexing. Cultures were then layered on a 75% Percoll (GE Healthcare, Chicago, IL) density gradient and centrifuging at 4000 rpm (3100 x g) for 15 min. The schizont layer (layer immediately above the Percoll) was harvested and washed once with RPMI hematocrit for 3 h. Then, 0-3 hpi rings were obtained following an additional treatment with 5% sorbitol.
To obtain a higher protein yield for Western blot experiments, early rings were obtained as described in 86 . Brie y, cultures were treated with 5% sorbitol a total of three times. Cultures were incubated 12 h between rst and second treatments, and then 36 h between second and third treatments. Trophozoite stage parasites (26-30 hpi) were obtained using two treatments with 5% sorbitol 12 h apart. Following the second treatment, parasites were cultured for an additional 12 h.
Drug treatments and lysate preparation Parasites were synchronized as described above and treated with the indicated compound for the indicated time under hypoxic conditions. DMSO concentration did not exceed 0.2%. Parasites were released from RBCs using 0.15% saponin (Acros Organics) then washed three times with 1 x PBS at 4°C. For Western blots, parasites were lysed with 1% Triton X-100 (Thermo Fisher Scienti c), 5% glycerol other primary antibodies were obtained from Cell Signaling Technologies (Danvers, MA). All secondary antibodies were obtained from Invitrogen (Waltham, MA). After washing 4 times with 1x TBS-T, blots were visualized using Immobilon Western Chemiluminescent HRP substate (Millipore Sigma). Blots were stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scienti c) between antibodies of the same species. Densitometry was performed with ImageJ version 1.53K. Statistical signi cance was analyzed with GraphPad version 9 using a two-tailed paired t-test.
Readings were taken every 3 min for 2 h or until uorescence exceeded the detection maxima. To determine activity, relative uorescence was plotted over time and the slope of the line was determined in Microsoft Excel. At least 3 biological replicates were performed for each substrate. Student t-tests were used to determine differences in relative activity.

Competition assays
Prior to starting competition assays, 159-2 parasites were grown in media containing 2 µg/mL blasticidin for a minimum of 2 weeks to ensure that > 90% of parasites were EGFP positive. Blasticidin selection pressure was removed prior to and through the duration of competition assays. Parasites of interest were adjusted to 1% parasitemia, and mixed 3:1 with the 159-2 parasite strain. A 1:1 ratio was not used since an initial experiment revealed that 159-2 parasites outcompete Cam3.II parasites within one week. Parasites were cultured in drug-free media at 5% hematocrit and maintained between 0.2 and 7% parasitemia. As a control, wells containing Cam3.II Kelch13 C580Y alone or 159-2 alone were grown concurrently to control for background EGFP uorescence or loss of EGFP expression in the absence of blasticidin, respectively. No loss in EGFP expression was noted in 159-2 parasites grown in the absence of blasticidin through the duration of competition assays.     with DMSO or 700 nM DHA for 3 h, then lysates were subject to Western blot and immunoblotted with antibodies against p-eIF2α and eIF2α. Shown is a representative blot from three independent experiments (see Supplementary Fig. 2a for replicates). (c) Densitometry analyses was performed using Image J and UPR activation determined as described in Fig.1. (d) WT and R539T parasites were synchronized to 0-3 hpi rings and treated with DMSO or 700 nM DHA for 3 h. Then drug was washed off and parasites were harvested at the indicated times to monitor UPR resolution. Western blot was performed as described in  Suc-LLVY-AMC to assess β1, β2, and β5 activity, respectively. Fluorescence was plotted over time and % activity was quanti ed by calculating the slope of the line and normalizing to the slope of DMSO-treated parasites. Bar graphs indicate mean % activity ± S.E.M. A two-tailed Student's t-test was performed between DMSO and drug-treatment counterparts, and statistical signi cance is indicated above the bars as vertical asterisks. Comparisons were also performed between Kelch13 WT and Kelch13 mutants for each treatment condition, but no signi cant difference was found (only signi cant comparisons between WT and mutant parasites are denoted here). (d-f) Parasites were synchronized as described above but treated with DMSO, 300 nM OZ439, 3 µM OZ439, or 2.5 µM WLL for 3h. Then, protein was harvested and proteasome activity was assessed as described above. (g) WT, R539T, and C580Y parasites were treated with DMSO or 50 nM DHA for the indicated times. Lysates were subjected to Western blot and immunoblotted with antibodies against K48-linked ubiquitin and BiP. Shown is a representative blot of four independent experiments. (see Supplementary Fig. 3 for replicates). (h) Densitometry analyses was performed with Image J and levels of K48-linked ubiquitination was normalized to the loading control BiP.

Supplementary Files
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