Plasmodium falciparum heat shock protein 110

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Received 12 Jun 2012 | Accepted 15 Nov 2012 | Published 18 Dec 2012

One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.

DOI: 10.1038/ncomms2306

Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers

Vasant Muralidharan1,2,w, Anna Oksman1,2, Priya Pal1,2, Susan Lindquist1,3,4 & Daniel E. Goldberg1,2

1 Howard Hughes Medical Institute, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA. 2 Departments of Medicine and Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri 63110, USA. 3 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02139, USA. 4 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.

w Present address: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA. Correspondence and requests for materials should be addressed to D.E.G. (email: mailto:[email protected]

Web End [email protected] ).

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The deadly malaria parasite, P. falciparum, has a proteome that is replete with amino-acid repeats; the majority of these repeats consist of asparagine (Asn) residues1,2. One

in four proteins in the P. falciparum proteome have Asn repeat-rich sequences, comprising up to 83 Asn residues and an average size of 37 residues3. There can be multiple repeats in a given protein. The presence of Asn-rich sequences in proteins is known to increase their propensity for aggregate formation46. Formation of aggregates is enhanced under heat shock stress conditions owing to an increase in protein unfolding7,8. In a recent survey of prion proteins in yeast, numerous Asn-rich sequences were found to form aggregates4. Prion-like aggregates have been shown to be responsible for the inheritance of several phenotypes in yeast9,10, to have a functional biological role in bacteria11, to be important for persistence of synaptic facilitation12 and to be vital for antiviral innate immunity13. While such regulated aggregation of proteins is benign or benecial, unregulated aggregation of proteins can lead to cell death6,14. The malaria parasite is able to thrive with an Asn repeat-rich proteome in face of the periodic heat shock stress that is a hallmark of clinical malaria. Patients suffer recurrent bouts of fever, often exceeding 40 1C and lasting several hours at a time.

The ability of P. falciparum to survive this insult is likely owing to processes that mask Asn repeat-rich protein aggregation.

Certain chaperones, particularly heat shock proteins (Hsps), have been shown to have a vital role in controlling aggregate formation15,16. In vitro, these chaperones act in concert to refold proteins and unfold preformed aggregates17. The Hsp70 Hsp110Hsp40 network refolds proteins via repeated cycles of binding and release of unfolded proteins by Hsp70 that is governed by its ADP or ATP bound state, respectively15,16. Hsp110 acts as a nucleotide exchange factor for Hsp70, exchanging ADP for ATP and thereby completing the refolding cycle1821. Hsp110 can also bind unfolded proteins, via its substrate-binding domain, but the role of this binding event in the refolding cycle is unclear2224 and its biological function remains undened. Recently, mammalian Hsp110 was found to localize with aggregates in human embryonic kidney (HEK) 293T cells but the signicance of the association is not understood14. Drosophila Hsp110 was identied in a genome-wide RNA interference screen as a mitigating factor for aggregation of Huntingtin protein25. This chaperone system clearly has a role in unfolded protein handling, but its capacity is easily overwhelmed by the expression of aggregation-prone proteins, especially under heat shock conditions. Heat shock stress increases the propensity of proteins to unfold, enhancing the formation of aggregates7,8.

We show here that the cytoplasmic Hsp110 of P. falciparum (PfHsp110c) is able to prevent aggregation of Asn repeat-rich proteins in cultured malaria parasites and in mammalian cells. The P. falciparum chaperone is much better at this than orthologs from yeast or humans. We conclude that PfHsp110c, in particular its substrate-binding activity, is vital for proteostasis of the Asn repeat-rich proteome of P. falciparum and propose that its presence allows the propagation of these repeats within the parasite proteome.

ResultsA P. falciparum Asn-rich protein aggregates in human cells. Aggregation of Asn repeat-rich proteins has not been observed within P. falciparum and we have reported that the presence of these repeats within a protein does not seem to affect its cellular function or location, even under heat shock26. This suggested that the parasite chaperone network has adapted to mask the aggregation propensity of its Asn repeat-rich proteome or that the parasite Asn repeat-rich proteins are not capable of

aggregating even during febrile episodes. To distinguish between these possibilities, we transiently transfected HEK293T cells with a P. falciparum protein that contains a stretch of 83 Asn residues (PlasmoDB ID: PF3D7_0923500 or PFI1155w). For comparison, we transfected HEK293T cells with the N-terminal Asn/glutamine (Gln)-rich prion-forming domain (PrD, amino acids 1125) of the yeast protein Sup35 (Sup35PrD)9,27. Each was fused to a monomeric derivative of red uorescent protein (tagRFP-T)28 The transfected HEK293T cells were incubated at 40 1C for 6 h and then assessed by uorescence microscopy (Fig. 1a,b). Protein aggregation, observed as distinct uorescent foci, was seen in a large proportion of the HEK293T cells expressing either Asn-rich protein (Fig. 1a,b and Supplementary Fig. S1a). The uorescent fusion protein, tagRFP-T, expressed alone in HEK293T cells did not form aggregates (Supplementary Fig. S2).

PfHsp110c prevents protein aggregation in human cells. We tested the ability of Hsp110 proteins from yeast, human andP. falciparum to prevent aggregation. Two Hsp110 proteins inP. falciparum were identied via sequence homology to human and yeast Hsp110 proteins (Supplementary Fig. S3). One of theP. falciparum Hsp110 proteins (PF3D7_134200 or MAL13P1.540) has a predicted signal sequence as well as an endoplasmic reticulum retention signal (Supplementary Fig. S3). We focused our efforts on the cytoplasmic Hsp110 (PF3D7_0708800 or PF07_0033, referred to as PfHsp110c). Co-expression of a PfHsp110cgreen uorescent protein (GFP) fusion with PFI1155w or Sup35PrD in heat-shocked HEK293T cells gave uniform cytoplasmic distribution of the Asn-rich proteins (Fig. 1c,d). In contrast, substituting orthologs from human (Hsp105a) or yeast (Sse1p or Sse2p) for PfHsp110c allowed substantial formation of uorescent foci in co-transfected cells (Supplementary Fig. S1bg). All fusion proteins were expressed at comparable levels in HEK293T cells (Supplementary Fig. S1h,i).

The formation of protein aggregates was quantied (Fig. 1e and Supplementary Fig. S1a). While Hsp105a, Sse1p and Sse2p co-expression gave 23-fold reductions in the appearance of Sup35PrD or PFI1155w protein aggregates after heat shock, co-expression of PfHsp110c reduced the formation of protein aggregates by 1015-fold compared with Asn-rich protein fusion construct alone (Fig. 1e). We conclude that Hsp110 homologues can reduce aggregation of Asn-rich proteins, and that PfHsp110c is substantially better at doing so than its yeast and human counterparts.

To determine if PfHsp110c is selective for Asn-rich proteins, all Asn or all Gln versions of Sup35PrD5, in which every Gln residue in Sup35PrD was changed to Asn or every Asn residue in Sup35PrD was changed to Gln, were substituted for the wild-type Sup35PrD in the HEK293T transfection experiment. PfHsp110c protected against aggregation of both versions and to a greater extent than its homologues in all cases (Fig. 1f). PfHsp110c appears to have a general aggregation protection function.

PfHsp110c is an essential gene. To investigate the role of PfHsp110c in the intraerythrocytic life cycle of the malaria parasite, we attempted to knock out the PfHsp110c gene using a double-crossover homologous recombination approach29 (Fig. 2a). Successful double-crossover integration of the hDHFR drug resistance cassette into the PfHsp110c gene was observed in all clones isolated from independent transfections (Supplementary Fig. S4). However, in the Southern blot probed with the 50-homologous region utilized for generating the knockout, we observed a second band corresponding to an uninterrupted endogenous gene (Fig. 2b). The data suggest that

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PFI1155wtagRFP-T

Sup35PrDtagRFP-T

Pf Hsp110cGFP + PFI1155wtagRFP-T Pf Hsp110cGFP + Sup35PrDtagRFP-T

Fold decrease in aggregation

Sup35PrD + Hsp105

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Sup35 AIIQ + Hsp105

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Sup35 AIIN + Sse2pSup35 AIIN + PfHsp110c

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Sup35 AIIQ + Sse1p

Sup35 AIIQ + Sse2p

Figure 1 | PfHsp110c prevents aggregation of Asn-rich proteins in heat-shocked mammalian cells. (a,b) The Asn repeat-rich parasite protein, PFI1155w (a), or the prion-forming domain of Sup35PrD (b) was fused to tagRFP-T and expressed in HEK293T cells. The cells were xed and observed by uorescence microscopy. The arrowheads point to uorescent foci of tagRFP-T fusion protein, indicating aggregation. Images from left to right are phase, 40,6-diamidino-2-phenylindole (DAPI), RFP, uorescence merge and all merge. Scale bar represents 10 mm. (c,d) PfHsp110cGFP was co-expressed with

PFI1155wtagRFP-T (c) or Sup35PrDtagRFP-T (d) in HEK293T cells. The cells were treated as in (a). Scale bars as in (a). Images from left to right are phase, DAPI, RFP, GFP and RFPGFP merge. (e,f) The reduction in aggregate formation as observed by blinded quantication of uorescent foci of tagRFP-T fusions for cells co-expressing Hsp110GFP fusions (Supplementary Fig. S1). The y axis represents the ratio of the fraction of cells showing uorescent foci that express the Asn- or Gln-rich tagRFP-T fusion protein alone to the fraction of cells showing uorescent foci that co-express Hsp110 proteins with the Asn- or Gln-rich tagRFP-T fusion protein. Greater than 300 double-transfected cells were assessed under each condition. Data are represented as means.e.m. (nZ3). The raw data are shown in Supplementary Fig. S1a.

the PfHsp110c gene underwent a duplication event and the hDHFR selection cassette integrated into one copy. Locus mapping showed that duplication was limited to the PfHsp110c gene and did not extend to neighbouring genes (Supplementary Fig. S4c). The inability to obtain disruptants of the PfHsp110c gene without gene duplication suggests an essential function for this gene30.

The study of essential genes in the haploid malaria parasite has been enhanced by degradation-domain-based conditional expression systems3133. We recently reported the use of a regulated uorescent afnity (RFA) tag based on the Escherichia coli DHFR degradation domain (DDD)26. In the absence of the folate analogue trimethoprim (TMP), the fusion protein is

unstable and thus targeted for degradation by the proteasome. In the presence of TMP, the fusion protein is stabilized and able to carry out its normal biological function. The PfHsp110c gene was RFA-tagged via a single-crossover homologous recombination strategy (Fig. 2c,d). Clones isolated from independent transfections were used for further analysis (1F9 and 2D8).

Growth of PfHsp110cRFA parasite lines in the absence or presence of 10 mM TMP was monitored over several days by ow cytometry (Fig. 3a). PfHsp110cRFA parasites did not grow in the absence of TMP and by 48 h no evidence of live parasites was seen by microscopy. Growth of PfHsp110cRFA parasite lines was TMP concentration dependant (Fig. 3b). We conclude that

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Genomic locus Genomic locus9.5 kb 11.3 kb

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Figure 2 | Knockout of PfHsp110c results in gene duplication. (a) Scheme showing the strategy utilized to knock out the PfHsp110c gene via double-crossover homologous recombination. Plasmid (p110-KO-TK) containing a positive drug selection marker, human dihydrofolate reductase (hDHFR), and a negative drug selection marker, thymidine kinase (TK), was transfected into the parent strain, 3D7. EcoRV restriction sites used to detect integration along with the expected sizes are indicated. (b) Southern blot of genomic DNA digested with EcoRV and probed with the 50-region of the PfHsp110c gene utilized for homologous recombination. The bands expected for double-crossover integration of the 50-homologous region were seen in all clones (*). The band expected for the undisrupted PfHsp110c gene in the parental line, 3D7, was also seen in all clones (grey). (c) Scheme showing the strategy utilized to fuse the RFA-tag to the 30-end of the PfHsp110c gene via single-crossover homologous recombination. Plasmid (p110-GDB) containing the blasticidin (BSD)

resistance cassette, for positive drug selection, was transfected into the parental strain, PM1KO. BbsI restriction sites along with the probe used to detect integration and the expected sizes are indicated. (d) Southern blots of genomic and plasmid DNA digested with BbsI. Bands expected from a single-crossover integration of the RFA-tag into the 30-end of the PfHsp110c gene were observed in both clones, 1F9 and 2D8, isolated from the two independent transfections (red). The band expected for the digested plasmid was also seen in both clones (black), suggesting that a plasmid concatamer integrated into the gene, a common occurrence in P. falciparum. A single band was observed for the parental strain (PM1KO, blue) that was absent in the integrant clones.

PfHsp110c provides to blood-stage parasites an essential function that is disrupted upon TMP removal owing to destabilization of the RFA tag.

PfHsp110c is required for surviving heat shock. High fever, which occurs periodically in malaria patients, is a cellular stress that results in a global tendency towards unfolding of proteins7,8. Asynchronous PfHsp110cRFA parasites were incubated at 40 1C for 46 h with or without TMP (Fig. 3c). After the heat shock, the parasites were transferred back into medium containing TMP and incubated at the normal growth temperature, 37 1C (Fig. 3c). The recovery of PfHsp110cRFA parasites was monitored over several days. In the absence of TMP, even a brief 4-h incubation at 40 1C severely inhibited parasite viability (Fig. 3c). In contrast, parasites incubated at 37 1C for 6 h without TMP were fully viable (Supplementary Fig. S5). Indeed, cultures kept at 37 1C without

TMP took 2 days to lose viability (Fig. 3a). The results demonstrate that PfHsp110c is vital for the parasite to survive even brief febrile temperatures, suggesting a role for PfHsp110c in the proteostasis of the Asn repeat-rich parasite proteome.

TMP controls PfHsp110cRFA function. We assessed protein levels in PfHsp110cRFA parasites. Asynchronous PfHsp110c RFA parasites were incubated at 37 or 40 1C in varying amounts of TMP for 24 h. Protein levels were monitored by western blots immunoprobed for GFP, which is a component of the RFA-tag (Supplementary Fig. S6a). Surprisingly, there was no TMP dose-dependent variation in PfHsp110cRFA levels at either temperature (Supplementary Fig. S6a). In addition, parasites incubated in the absence of TMP showed no time-dependent degradation at 37 or 40 1C (Supplementary Fig. S6b). In contrast, we have seen robust degradation of other RFA-tagged proteins26.

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25 1F9 + TMP 1F9 TMP 2D8 + TMP 2D8 TMP

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Figure 3 | PfHsp110c is essential and is required for surviving febrile temperatures. (a) Asynchronous PfHsp110cRFA parasite clones, 1F9 and 2D8, were grown with or without 10 mM TMP and their growth was monitored over 5 days via ow cytometry. Data are t to an exponential growth equation and are represented as means.e.m. (n 3). (b) Asynchronous PfHsp110cRFA parasite clones, 1F9 and 2D8, were incubated in different TMP concentrations and

their growth was monitored after 3 days by ow cytometry. Data are t to a doseresponse equation and are represented as means.e.m. (n 3).

(c) Asynchronous PfHsp110cRFA parasite clones, 1F9 and 2D8, were subjected to heat shock using the protocol shown, permissive conditions were restored and their growth was monitored over 3 days by ow cytometry. Data are t to an exponential growth equation and are represented as means.e.m. (n 3). For each experiment, parasites were subcultured with fresh erythrocytes at day 3 and parasitemia is calculated accounting for culture

dilution. Media were changed every day.

As Hsp110 proteins act in concert with Hsp70 and their nucleotide exchange factor activity is required for Hsp70 function1820,24, we hypothesized that in the absence of TMP the RFA-tag interferes with essential PfHsp110c interactions resulting in parasite death. We tested this model using co-immunoprecipitation experiments. PfHsp110cRFA parasites were incubated at 37 or 40 1C with or without TMP for 6 h as in

Fig. 2c. Total PfHsp110cRFA and PfHsp70 levels in lysates were stable and no degradation of PfHsp110cRFA was observed in the absence of TMP (Fig. 4a). When PfHsp70 was immunoprecipitated from the lysates, higher amounts of PfHsp110cRFA co-precipitated at 40 1C compared with 37 1C (Fig. 4b) indicating that more Hsp70Hsp110 complex exists at the higher temperature. However, there was no TMP-dependent difference in the amount of co-precipitated PfHsp110cRFA at either temperature. We assessed the reciprocal interaction by immunoprecipitating PfHsp110cRFA using an anti-GFP monoclonal antibody, 3E6 (Fig. 4c), and western blotting with a second anti-GFP monoclonal antibody, JL8. The amounts of PfHsp110cRFA and of co-precipitated PfHsp70 did not vary at 37 1C with or without TMP. However, at 40 1C, less PfHsp110c

RFA was immunoprecipitated in the absence of TMP and correspondingly lower amounts of PfHsp70 were seen (Fig. 4c). The data show that at 40 1C without TMP, the 3E6 monoclonal anti-GFP antibody used for immunoprecipitation of PfHsp110c RFA is unable to recognize the GFP epitope within the RFA-tag, even though the protein is clearly in the lysates (Fig. 4a).

Similar results were obtained using a second approach, semi-denaturing detergent agarose gel electrophoresis (SDDAGE)34,35, and another anti-GFP antibody (Supplementary Fig. S7). The co-immunoprecipitation and SDD-AGE data suggest that the unfolded DDD within the PfHsp110cRFA is occluding the GFP within the RFA-tag by binding intramolecularly to the substrate-binding domain of PfHsp110c.

PfHsp110c and Thioavin T rescue the knockdown phenotype. PfHsp110cRFA parasites die upon TMP removal even though PfHsp110cRFA protein levels do not decrease. To test if the death of PfHsp110cRFA parasites is owing to the absence of chaperone function or owing to fusion protein toxicity, we episomally expressed PfHsp110c fused to tagRFP-T, under the control of the native PfHsp110c promoter in PfHsp110cRFA parasites. The complemented PfHsp110cRFA parasites were subjected to heat shock as in Fig. 2c. Parasites complemented with PfHsp110ctagRFP-T but not with tagRFP-T alone were able to recover after the heat shock in the absence of TMP (Fig. 5a). The recovery is partial because plasmid maintenance is incomplete (data not shown). In contrast, PfHsp110 RFA parasites expressing either the human Hsp105a or the yeast Hsp110 homologues (Sse1p and Sse2p) under the control of the PfHsp110c promoter were unable to recover after heat shock in the absence of TMP (Fig. 5b). These ndings support our model that the death of PfHsp110cRFA parasites

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1F9

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Figure 4 | Function of PfHsp110cRFA is controlled by TMP.(ac) PfHsp110cRFA parasites were incubated at 37 or 40 1C with or without 10 mM TMP for 6 h (indicated at top). PfHsp110cRFA (using monoclonal aGFP JL8) and PfHsp70 (using polyclonal aHsp70) were detected by immunoblotting after SDSPAGE. (a) Total cell lysates,(b) immunoprecipitation of PfHsp70 using polyclonal aHsp70 and(c) immunoprecipitation of PfHsp110cRFA using monoclonal aGFP 3E6.

Parasite clones 1F9 and 2D8 are indicated at the top.

0 1 2 3 4 5

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after 5 days (%)

in the absence of TMP is specically owing to the loss of PfHsp110c function.

Thioavin T (ThT) has been utilized to monitor protein aggregation in vitro36. Recently, ThT was successfully used to maintain proteostasis and extend the life span of Caenorhabditis elegans expressing a polyglutamine protein37. PfHsp110cRFA parasites were incubated at 40 1C for 6 h in the absence of TMP and in the presence or absence of 100 nM ThT and then allowed to recover. There was substantial and reproducible rescue of parasite growth (Fig. 5c). Higher ThT concentrations had toxic effects independent of TMP. This result suggests that in the absence of a functional PfHsp110c, parasites die owing to disruption of proteostasis in its Asn repeat-rich proteome.

Sup35PrD and PFI1155w expression in PfHsp110cRFA parasites. To independently test this idea, we episomally expressed PFI1155wtagRFP-T and Sup35PrDtagRFP-T in PfHsp110cRFA parasite clones. Transfected parasites were incubated at 40 1C for 6 h with or without TMP (Fig. 6). Those subjected to heat shock in the presence of TMP showed uniform cytoplasmic distribution of PFI1155wtagRFP-T (Fig. 6a) and Sup35PrDtagRFP-T (Fig. 6c), as well as PfHsp110cRFA (in green). However, parasites that were subjected to heat shock without TMP showed foci of PFI1155wtagRFP-T (Fig. 6b) and Sup35PrDtagRFP-T (Fig. 6d) uorescence indicative of protein aggregation9,27. The distribution of PfHsp110cRFA also was more focal upon heat shock in the absence of TMP (Fig. 6b,d)

1F9 TMP

1F9 TMP + 100 nM ThT

2D8 TMP

2D8 TMP + 100 nM ThT

Figure 5 | The knockdown phenotype is specic and rescued only by PfHsp110c. (a) Parasites were complemented with PfHsp110ctagRFP-T or tagRFP-T. Asynchronous complemented parasites were incubated at 40 1C for 6 h with or without 10mM TMP as in Fig. 3c. Their recovery was monitored over 5 days by ow cytometry. Data were t to an exponential growth equation and are represented as means.e.m. (n 3). (b) Parasites were complemented

with PfHsp110ctagRFP-T, human Hsp105atagRFP-T, yeast Sse1ptagRFP-T, Sse2ptagRFP-T or tagRFP-T. Asynchronous complemented parasites were incubated at 401C for 6 h without TMP. Their recovery post heat shock after 5 days was normalized to that of complemented PfHsp110cRFA parasites that were heat shocked in the presence of 10 mM TMP. Parasitemia was monitored daily via ow cytometry. Data are represented as means.e.m. (n 3).

(c) Parasites were incubated at 401C for 6 h without TMP with or without 100nM ThT. Their recovery after 5 days was normalized to parasites incubated at 401C for 6 h with 10mM TMP and with or without 100nM ThT. Data are represented as means.e.m. (n 3).

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Pf Hsp110cRFA + PFI1155wtagRFP-T with TMP at 40 C, 6 h Pf Hsp110cRFA + PFI1155wtagRFP-T without TMP at 40 C, 6 h

Pf Hsp110cRFA + Sup35PrDtagRFP-T with TMP at 40 C, 6 h Pf Hsp110cRFA + Sup35PrDtagRFP-T without TMP at 40 C, 6 h

Figure 6 | PfHsp110c prevents the aggregation of Asn-rich protein during febrile episodes. Live uorescence images of PfHsp110cRFA parasite lines expressing (a,b) the Asn repeat-rich parasite protein, PFI1155w, or (c,d) the prion-forming domain from the yeast protein, Sup35 (aa1-125), fused to tagRFP-T (Sup35tagRFP-T), incubated at 40 1C for 6 h with (a,c) or without (b,d) 10 mM TMP. The arrowheads indicate uorescent foci of PFI1155wtagRFP-T (b) and Sup35tagRFP-T (d). Scale bar represents 2 mm. Images from left to right are phase, DAPI, GFP, RFP and GFPRFP merge.

and there was minimal overlap between either PFI1155w tagRFP-T or Sup35PrDtagRFP-T and PfHsp110cRFA foci (Fig. 6b,d). Expression of tagRFP-T alone in PfHsp110cRFA lines gave diffuse cytoplasmic uorescence in all conditions (Supplementary Fig. S8), showing that aggregation was dependent on the Asn-rich sequence. These observations support our model that PfHsp110c has an essential role in preventing parasite Asn-rich protein aggregation.

DiscussionThe reason for the widespread presence of Asn repeat-rich proteins in the proteome of the deadly malaria parasite,P. falciparum, remains a mystery. Indeed, proteins with large Asn-rich regions are prone to forming aggregates and prion-like brils46 and thus might be expected to reduce the tness of malaria parasites. Furthermore, the propensity of Asn-rich proteins to aggregate is enhanced by elevated temperatures7,8, a scenario that P. falciparum parasites routinely encounter during febrile episodes that are a hallmark clinical manifestation of malaria. In this study, we have uncovered a central role of PfHsp110c in maintaining proteostasis and preventing aggregation of the Asn repeat-rich proteome of P. falciparum (Fig. 7). This chaperone is essential for parasite growth within the red blood cell (Figs 2 and 3) and its substrate-binding activity is necessary for surviving even a brief exposure to febrile

temperatures (Figs 3 and 4 and Supplementary Fig. S7). The sequence homology of PfHsp110c to human and yeast Hsp110 proteins is only about 24%, with most of the homology in the nucleotide-binding region (Supplementary Fig. S3). The divergent susbtrate-binding region of PfHsp110c could be responsible for the better aggregation prevention properties of the P. falciparum homologue.

The malaria parasite has to deal with regular exposure to temperatures of 40 1C (during febrile episodes). We therefore tested the ability of PfHsp110cRFA parasites to survive heat shock in the absence of the stabilizing ligand (Fig. 3c). The PfHsp110cRFA parasites were killed within a few hours, faster than killing achieved by most antimalarial drugs. However, when we assessed cellular levels, we found that unlike other RFA-tagged parasite proteins26, PfHsp110cRFA is not degraded in the absence of TMP (Fig. 4a and Supplementary Fig. S6). PfHsp110cRFA parasite lines were able to recover from heat shock when they were complemented with PfHsp110c but not with the human or yeast Hsp110 homologues, underscoring the specicity of the phenotype (Fig. 5a,b). By using monoclonal antibodies against the GFP and HA modules of the RFA tag under more or less denaturing conditions, we were able to show that the unfolded degradation domain within the RFA-tag was occluding the epitopes of two anti-GFP monoclonal antibodies when PfHsp110cRFA parasites were heat shocked (Fig. 4b,c and Supplementary Fig. S7). Presumably, PfHsp110cRFA was busy

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Folded protein

Heat shock

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Mis/unfolded protein

Folded protein

Hsp70Hsp110 Hsp40 cycle

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Hsp110 substrate binding domain

RFA tag

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DDDHA

GFP

DDDHA

Drug

GFP

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Folded DDD

DDDHA

+ Drug

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Hsp Hsp

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Figure 7 | Proposed role of Hsp110 in maintaining an aggregate-free Asn repeat-rich proteome. Proteins containing Asn-rich regions are misfolded or unfolded during heat shock. The misfolded proteins form aggregates in human and yeast cells. However, in the malaria parasite, despite an abundance of Asn repeat-rich proteins, there is no protein aggregation. The prevention of protein aggregation in P. falciparum depends upon the activity of PfHsp110c that acts in a network with other chaperones (Hsp70 and other Hsp). PfHsp110cRFA function is dependent upon the stabilizing ligand, TMP. When TMP is present, Hsp110 can productively function in the Hsp70Hsp110Hsp40 cycle resulting in a stable Asn repeat-rich proteome. In the absence of TMP, the DDD within the RFA tag is unfolded. The unfolded DDD fused to Hsp110 results in the inhibition of chaperone activity. This leads to protein aggregation in the Asn repeat-rich proteome. The Hsp110 and Hsp70 structures (PDB:3C7N) were rendered using PyMol.

binding its own tag, preventing it from carrying out its usual role in binding other aggregation-prone, heat-shocked proteins and thus allowing the use of TMP to modulate chaperone function (Fig. 7). Utilizing degradation domains whose unfolding is controlled by small molecules could be a general technique to manipulate chaperone function in vivo. Our experiments failed to detect any TMP-rescued inactivation of PfHsp110cRFA function at 37 1C (Fig. 4b,c and Supplementary Fig. S6). We believe that because death of PfHsp110cRFA parasites upon removal of TMP at 37 1C takes much longer (2 days) than at 40 1C (46 h)

(Fig. 3a,c), the imbalance in proteostasis is more subtle at 37 1C.

Does PfHsp110c affect proteostasis of the Asn repeat-rich parasite proteome? We investigated this question in two ways: by testing the ability of the aggregate-binding small molecule, ThT, to rescue the growth of PfHsp110cRFA parasites heat shocked in the absence of TMP (Fig. 5c) and by expressing Asn repeat-rich proteins (PFI1155w and Sup35PrD), in PfHsp110cRFA parasites (Fig. 6). In PfHsp110cRFA clones, ThT was able to substantially rescue parasite viability (Fig. 5c). In addition, expression of PFI1155w or Sup35PrD in P. falciparum did not lead to formation of aggregates (Fig. 6a,c), in contrast to what happens in yeast9,27 and in mammalian cells (Fig. 1a,b). Only in the absence of TMP (functional knockdown of PfHsp110c) and after heat shock did aggregates of PFI1155w and Sup35PrD form (Fig. 6b,d).

In HEK293T cells, Hsp110 orthologs from multiple organisms were able to modulate aggregation of the Asn-rich proteins Sup35PrD and PFI1155w but PfHsp110c did so to a much greater

extent (Fig. 1). PfHsp110c prevented aggregation of not only Asn-rich proteins but also of a Gln-only version of the Sup35PrD (Fig. 1f), suggesting that its ability to handle aggregates does not depend on the sequence of the aggregating protein. These ndings show that P. falciparum has evolved an Hsp110-dependent aggregation-resistance mechanism (Fig. 7). We propose that PfHsp110c may function as a cellular capacitor38, allowing the rampant expansion of Asn repeats in surface loops, where tolerated1. These repeats could then evolve over time into new functional domains of advantage to the organism.

We have demonstrated that PfHsp110c is essential for parasite survival within the host red blood cell. We have also shown that PfHsp110c is vital for maintaining proteostasis in P. falciparum (Fig. 7). In fact, PfHsp110c is able to prevent aggregation even in HEK293T cells, suggesting that its interacting partners in the chaperone network can be interchanged with distant orthologs. The identication of other chaperones that act in concert with PfHsp110c and their roles in maintaining the proteostasis of the Asn repeat-rich parasite proteome are active areas of future research. The ability to hamper the proteostasis of the imbalanced parasite proteome by inhibiting PfHsp110c function should make it an attractive target for drug development. Our ndings also raise the intriguing possibility of utilizing PfHsp110c to prevent protein-misfolding diseases.

Methods

DNA sequences and cloning. Genomic DNAs were isolated from P. falciparum using the Qiagen Blood and Cell Culture kit. Primers and restriction sites (New England Biolabs) used in this study are listed in Supplementary Table S1. All constructs utilized in this study were conrmed by sequencing. All PCR products were inserted into the respective plasmids using the In-Fusion cloning system (Clonetech). The p110-KO-TK vector was derived from pHHT-TK vector29.

A 1-kb homologous sequence from the 50-end of the PfHsp110c gene and a 1-kb homologous sequence from the 30-end of the PfHsp110c gene were amplied by PCR using primers in Supplementary Table S1. The 50-homologous region was introduced using SacII and BglII (New England Biolabs) restriction sites and the 30-homologous region was introduced using ClaI and NcoI (New England Biolabs)

restriction sites into the pHHT-TK vector, anking the hDHFR cassette, to make p110-KO-TK vector. Episomal vectors for parasites were constructed from the plasmid containing Plasmepsin II in-frame with GFP (pPM2GT)39 by inserting a PCR product (Supplementary Table S1) comprising the hsp86 or the hsp110 promoter using AatII and XhoI (New England Biolabs) restriction sites. These vectors were further modied by replacing the hDHFR drug selection cassette with the yeast dihydroorotate dehydrogenase selection cassette40 using the BamH1 (New England Biolabs) restriction site (plasmid kindly provided by Eva Istvan). The GFP was replaced with the PCR product containing the uorescent protein, tagRFP-T (Supplementary Table S1), that was introduced into these vectors using AvrII and EagI (New England Biolabs) restriction sites. For expressing tagRFP-T alone, the PCR product comprising tagRFP-T (Supplementary Table S1) was introduced into the vector using XhoI and EagI (New England Biolabs) restriction sites. The PCR products comprising Sup35PrD or PFI1155w (Supplementary Table S1) were introduced, in-frame with tagRFP-T, into the episomal vector with the hsp86 promoter using XhoI and AvrII restriction sites. The PfHsp110c, human Hsp105a and the two yeast Hsp110s (Sse1p and Sse2p) open reading frame (ORF) PCR products (Supplementary Table S1) were introduced, in-frame with tagRFP-T, into the episomal vector containing the hsp110 promoter using XhoI and AvrII restriction sites. The PCR products comprising Sup35PrD or PFI1155w (Supplementary Table S1) were inserted into pcDNA3.1 in-frame with tagRFP-T using HindIII and BamHI (New England Biolabs) restriction sites. The PCR product PfHsp110c ORF in-frame with GFP (Supplementary Table S1) was introduced into pLexm vector using NotI and XhoI (New England Biolabs) restriction sites. The PfHsp110c ORF from this modied pLexm vector was replaced, in-frame with GFP, with PCR products comprising human Hsp105a (Origene), Sse1p and Sse2p (Supplementary Table S1) using NotI and AvrII restriction sites.

Cell culture and transfections. 3D7 parasites were cultured in RPMI medium supplemented with Albumax and transfected as described earlier41,42. Parasites transfected with p110-KO-TK underwent positive selection at 48 h with 10 nM WR99210. After parasites appeared from WR99210 selection, they were cycled off drug for 3 weeks. Double-crossover integrants were then selected by applying10 nM WR99210 and 20 mM ganciclovir. Plasmepsin I-knockout parasites26,43 transfected with p110-GDB underwent positive selection 48 h after transfection with 2.5 mg ml 1 BSD (Calbiochem) and 10 mM TMP (Sigma). Integration was

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detected after two rounds of BSD cycling. Parasites were always cultured with10 mM TMP after it was initially introduced into the medium. In all cases, clones were isolated via limiting dilution. PfHsp110cRFA parasites were also transfected with episomal vectors that contained a yeast dihydroorotate dehydrogenase selection marker40 allowing selection for parasites maintaining the episome using 2 mM DSM-1. For growth curves, parasites were washed twice and incubated in the required medium and temperature. Medium was changed everyday and parasites were subcultured with fresh red blood cells every 3 days. Human kidney cell line 293T (HEK293T) was maintained as recommended by the ATCC. HEK293T cells were transfected using the X-tremeGENE 9 DNA Transfection Reagent (Roche) as per the manufacturers instructions. After 40-h incubation with transfection reagent and plasmid DNA, cells were heat shocked for 6 h and xed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS to be analysed by microscopy.

Southern blot. Southern blots were performed with genomic DNA isolated using the Qiagen Blood and Cell Culture kit. For PfHsp110c-knockout parasites, 1 mg of

DNA was digested overnight with EcoRV (New England Biolabs) and integrants were screened using probes against the positive selection marker, hDHFR, and the 50-homologous region used for integration. For PfHsp110cRFA parasites, 1 mg of

DNA was digested overnight with BbsI (New England Biolabs) and integrants were screened using probes against the 30-end of the PfHsp110c ORF. All Southern blots were performed as described earlier39.

Co-immunoprecipitation. Immunoprecipitation was carried out as described earlier26. The soluble fraction of the parasite lysates were incubated for 1 h with0.2 mg mouse monoclonal anti-GFP, 3E6 (Invitrogen), to immunoprecipitate PfHsp110cRFA or 0.5 mg rabbit polyclonal anti-Hsp70 (Agrisera) to immunoprecipitate PfHsp70 and 50 ml of Protein G-linked Dynabeads (Invitrogen). The beads were then washed four times with PBS containing protease inhibitor cocktail (Roche). The washed beads were solubilized in SDSPAGE loading buffer (LICOR Biosciences) and fractionated by 10% SDSPAGE to be analysed by western blot.

Western blot and SDD-AGE. Western blots were performed as described previously26. For SDD-AGE, parasites were collected and host red blood cells were permeabilized selectively by treatment with ice-cold 0.04% saponin in PBS for10 min, followed by a wash in ice-cold PBS. Lysates from 5 107 cells were loaded

per lane. SDD-AGE was performed as described34. The antibodies used in this study were mouse monoclonal anti-GFP, JL8 (1:4000) (Clonetech), mouse monoclonal anti-HA, 3F10 (1:3000) (Roche), monoclonal anti-cyclinB1, GNS1 (1: 2500) (Santa Cruz), rabbit polyclonal anti-Hsp70 (1:3000) (Agrisera) and rabbit polyclonal anti-EF1a (1: 3000)44. The primary antibodies were detected using

IRDye 680CW (1: 15,000) conjugated goat anti-rabbit IgG (LICOR Biosciences and IRDye 800CW (1:15,000) conjugated goat anti-mouse IgG (LICOR Biosciences). The western blot images were processed and analysed using the Odyssey infrared imaging system software (LICOR Biosciences).

Flow cytometry. Aliquots of parasite cultures (5 ml) were stained with 1.5 mg ml 1 Acridine Orange (Molecular Probes) in PBS. The uorescence proles of infected erythrocytes were analysed by ow cytometry on a BD FACSCanto (BD Biosystems) or MACSQuant Analyser (Miltenyi Biotec). The parasitemia data were t to standard growth curve or doseresponse equations (nonlinear least-squares analysis) in the software package GraphPad Prism v.5.0a.

Microscopy. Live parasites were stained with 2 mM Hoechst 33342 (Molecular Probes) as described previously39. HEK293T cells were grown on coverslips pretreated with 0.01% poly-L-lysine (Sigma), xed and mounted on ProLong Gold with DAPI (Invitrogen), before microscopy. Cells were observed on an Axioscope Microscope (Carl Ziess Microimaging)42. Images were analysed and processed using ImageJ (National Institutes of Health) and merged images were generated using Adobe Photoshop.

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Acknowledgements

We thank Lauren Pepper for providing Sse1p and Sse2p and for helpful discussions, Heather True for providing Sup35 and anti-Sup35 antibody, Akhil Vaidya for providing DSM-1 and DHODH plasmid, Mike Diamond and Hyelim Cho for HEK293T cells, Roger Tsien for tagRFP-T, Niraj Tolia for pLexm, Barb Vaupel for technical assistance, Paul Sigala for comments on the manuscript, Andrey Shaw for helpful suggestions and the US National Institutes of Health (grant 1K99AI099156-01 to V.M.) for funding.

Author contributions

V.M. and D.E.G. designed the study, interpreted the results and wrote the manuscript.V.M., A.O. and P.P. performed the experiments and interpreted the results. S.L. contributed novel reagents before publication and helped interpret the results.

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How to cite this article: Muralidharan, V. et al. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat. Commun. 3:1310 doi: 10.1038/ncomms2306 (2012).

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Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers

Author

Muralidharan, Vasant; Oksman, Anna; Pal, Priya; Lindquist, Susan; Goldberg, Daniel E

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1310

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2012

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Dec 2012

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English

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https://doi.org/10.1038/ncomms2306

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1247513051

Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers

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