Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various “client” proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

Here, we show that a class of heat-soluble proteins, which we call “Hero” (heat-resistant obscure) proteins, are widespread in non-extremophilic animals such as Drosophila and humans. Hero proteins act as molecular shields to protect various “client” proteins from denaturation even under stress conditions, such as heat shock, desiccation, and exposure to organic solvents. Moreover, Hero proteins can suppress several forms of pathogenic protein aggregates in cells and in in vivo models for neurodegenerative diseases, and extend life span of Drosophila. Our findings not only have significant implications for our fundamental understanding of protein stability and functions but also highlight potential biotechnological and therapeutic applications of Hero proteins.

Proteins are polymers composed of 20 different amino acids, whose side chains have various properties such as aliphatic, aromatic, acidic, basic, and sulfur-containing. This diversity allows proteins to fold into three-dimensional structures, which determine their activity and function. Although proteins are generally stable at the physiological temperature or even at approximately 50–60°C [ 1 ], heating at near-boiling temperature destroys structures and induces denaturation and aggregation for most proteins. However, there are some exceptions. For example, tardigrade disordered proteins (TDPs), found in the supernatant of tardigrade lysate after boiling [ 2 ], are required for tardigrades to survive desiccation [ 3 ]. Another example is late embryogenic abundant (LEA) proteins in plants, whose expression is associated with their tolerance to dehydration, freezing, or high salinity [ 4 , 5 ]. LEA proteins are also found in extremophiles including the radiation-resistant bacterium Deinococcus radiodurans and desiccation-tolerant animals such as Artemia, Caenorhabditis elegans, and rotifers [ 6 , 7 ]. TDPs and most LEA proteins are extremely hydrophilic and heat soluble, and can be classified into intrinsically disordered proteins (IDPs). Currently, these heat-resistant proteins are viewed as special cases required by organisms living in extreme conditions to protect their functional proteins. Although mammals have been reported to produce some highly heat-soluble proteins [ 8 , 9 ], their functions remain largely unexplored.

Results

Boiled supernatants of crude cell lysates improve Ago2 molecular behavior Many proteins are naturally unstable even under physiological conditions, probably because they need to prioritize function over structural durability. For example, Argonaute (AGO) proteins, the core of RNA-induced silencing complex (RISC), must load a small RNA duplex, eject the passenger strand from the two strands of the duplex, recognize target RNAs complementary to the remaining guide strand, and endonucleolytically cleave RNA targets or recruit downstream silencing factors [10]. AGO proteins are highly unstable; in particular, empty AGO is structurally flexible at the single-molecule level [11] and susceptible to proteolytic digestion in vitro [12]. During the course of our studies on AGO, we observed a strange phenomenon: when immunopurifying Drosophila Ago2 fused with a tobacco etch virus (TEV)-cleavable FLAG-tag using anti-FLAG magnetic beads, we were unable to elute the protein from the beads, even after the FLAG-tag was removed using TEV protease (Fig 1A and 1B). We concluded that purified, free Ago2 protein is unstable and tends to stick nonspecifically to the beads. Strikingly, addition of crude Drosophila S2 cell lysate during the TEV protease treatment promoted efficient elution of Ago2 (Fig 1B). To test whether a protein(s) was responsible for this effect, we boiled the crude S2 cell lysate at 95°C for 15 minutes and removed the precipitated proteins by centrifugation. Unexpectedly, the supernatant of the heat-denatured lysate, which contained only approximately 4.3% of the total protein concentration in the original crude lysate, was as competent in eluting Ago2 as the original lysate. Digestion of proteins in the boiled supernatant with proteinase K, followed by its inactivation by a second round of heating, halved its activity in Ago2 elution (Fig 1B). To explore what materials other than proteins contribute to Ago2 elution, we utilized benzonase to deplete DNAs and RNAs. Treatment of the boiled lysate by both proteinase K and benzonase abolished the elution activity, while the benzonase treatment alone only modestly decreased the activity (Fig 1B). Elution of Drosophila Ago2 was similarly promoted by crude lysate from human HEK293T cells and by its boiled supernatant (approximately 6.0% of the original protein concentration). Again, the efficiency was nearly halved by the proteinase K treatment, modestly decreased by benzonase, and abrogated by proteinase K and benzonase together (Fig 1B). These results suggest that some heat-soluble proteins as well as nucleic acids contained in the boiled S2 or HEK293T cell supernatants promote elution of free Ago2 from the beads. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Activity of heat-soluble proteins and nucleonic acids on Ago2 molecular behavior and LDH desiccation. (A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody, and then the FLAG-tag was cleaved off by TEV protease in buffer, crude lysates, or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by benzonase. BZ/PK indicates that DNA and RNA had been degraded by benzonase, followed by the deproteinization by proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (bottom) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2 normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight, and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments. The numerical data pertaining to this figure can be found in S1 Data file. Ago, Argonaute; GST, glutathione S-transferase; Hsp, heat shock protein; LDH, lactate dehydrogenase; RISC, RNA-induced silencing complex; TEV, tobacco etch virus. https://doi.org/10.1371/journal.pbio.3000632.g001 We asked whether the boiled supernatants were capable of influencing any of the functional features of Ago2. Assembly of Drosophila Ago2-RISC requires not only a small RNA duplex and Ago2 itself but also the Dicer-2/R2D2 heterodimer and the heat shock protein (Hsp)70/90 chaperone machinery [13]. All these factors are present in crude S2 cell lysate, and incubation of FLAG-tagged Ago2 immobilized on magnetic beads with a 32P-radiolabeled small RNA duplex and S2 cell lysate efficiently assembled pre-RISC, containing both strands of the duplex, and mature RISC, containing only the single-stranded guide (Fig 1C). Compared to the crude S2 lysate, reconstitution of Ago2-RISC assembly using high concentrations of purified Hsp70/90 chaperone machinery and Dicer-2/R2D2 efficiently formed pre-Ago2-RISC, but its conversion into mature Ago2-RISC was inefficient. Adding the supernatant of heat-denatured S2 or HEK293T cell lysates strongly promoted the formation of both pre- and mature Ago2-RISC, although without the chaperones, the supernatants themselves showed no reconstitution activity (Fig 1C and 1D). Thus, the boiled supernatants from either fly S2 or human HEK293T cells can promote the assembly of Ago2-RISC by Dicer-2/R2D2 and the chaperone machinery. Together, our data suggest that factors in the boiled supernatants improve the molecular behavior of Ago2, either in its free form (Fig 1A and 1B) or upon RISC assembly (Fig 1C and 1D), and that these factors act beyond the species boundary.

Drosophila and human heat-soluble proteins protect lactate dehydrogenase activity from desiccation We next asked whether heat-soluble proteins in the boiled supernatants could act generally to enhance or protect the activity of other proteins, especially in stress conditions. We first examined the effect of boiled supernatants on protein dehydration. When purified lactate dehydrogenase (LDH) from rabbit muscle was dried up at room temperature overnight, addition of the boiled supernatants from S2 or HEK293T cells efficiently preserved the LDH activity compared to the PBS buffer only condition (Fig 1E). This effect was eliminated by proteinase K and/or benzonase treatment of the supernatants (Fig 1E), indicating that heat-soluble Drosophila and human proteins as well as nucleic acids can protect the activity of LDH from desiccation. Because it is already known that nucleic acids participate in maintaining protein homeostasis [14,15], we decided to focus on heat-soluble proteins in the following analyses.

Hero proteins protect the activity of various proteins under stress conditions To evaluate the function of each of the six representative Hero proteins, we produced recombinant human Hero proteins in Escherichia coli. We then mixed 5 μg/mL of LDH with 4 μg/mL of each Hero protein and subjected the mixture to overnight dehydration. Like the boiled supernatants (Fig 1E), all six Hero proteins protected the LDH activity at approximately 50%, which was much stronger compared to bovine serum albumin (BSA), GST, or the conventional protein stabilizers arginine or trehalose (Fig 3B). The LDH-protecting activity under desiccation is similar to what was previously observed for TDPs [3], which share no common sequence motifs with human Hero proteins. We next explored the protective effect of Hero proteins in organic solvents, a drastically harsh and foreign condition for proteins. When GFP was exposed to chloroform, its fluorescent intensity was reduced to approximately 10% of the native state (Fig 3C). In contrast, the addition of four out of six Hero proteins maintained >60% of the GFP activity (Fig 3C). Strikingly, in the presence of Hero45, GFP fluorescence remained undiminished even in chloroform (Fig 3C). This is reminiscent of the ability of methacrylate-based random heteropolymers (RHPs) to preserve horseradish peroxidase (HRP) activity in toluene [19]. Thus, naturally occurring Hero proteins act as molecular shields to stabilize proteins, even against harsh conditions. To investigate the effect of Hero proteins in cells, we expressed firefly luciferase together with each of the six Hero proteins, Hsp70, or Hsp90 in HEK293T cells, then subjected the cells to heat shock at 45°C for 8 minutes. Heat shock reduced the luciferase activity to between 10% and 20% of the original level when co-transfected with an empty or GST-expressing vector. Overexpression of the conventional chaperones, Hsp70 or Hsp90, maintained between 30% and 50% of luciferase activity (Fig 3D). Remarkably, Hero proteins protected luciferase from the heat shock as well as the chaperones, with Hero9, -7, and -11 preserving approximately 40% of luciferase activity (Fig 3D). We concluded that Hero proteins have the ability to stabilize various proteins both in vitro and in cells.

Hero proteins prevent pathogenic protein aggregations in cells Protein instability is often linked to diseases, especially neurodegenerative disorders. For example, aggregation of trans-activation response element (TAR) DNA-binding protein of 43 kDa (TDP-43) is observed in virtually all cases of amyotrophic lateral sclerosis (ALS) and in about half the cases of frontotemporal dementia (FTD) [20]. Given the strong activity of Hero proteins to stabilize proteins (Figs 1–3), we tested if Hero proteins could prevent pathogenic aggregation of TDP-43. We constructed TDP-43 lacking the nuclear localization signal (TDP-43ΔNLS), which is more prone to aggregation than the wild type because of its forced cytoplasmic localization. We expressed TDP-43ΔNLS as a GFP fusion in HEK293T cells, together with each of the six human Hero proteins or GST as a control. Aggregates of GFP-TDP-43ΔNLS and their suppression by co-expression of Hero proteins were apparent at the microscopic level (Fig 4A). To quantitatively analyze the degree of aggregation, the cells were lysed by sonication, and aggregates in the extracts were measured by a filter trap assay using a 0.2 μm cellulose acetate membrane in the presence of 1% SDS. We found that Hero45, Hero7, and Hero11 strongly suppressed TDP-43ΔNLS aggregation, compared to typical structured proteins such as GST, firefly luciferase (Luc), phosphoglycerate kinase 1 (PGK1), eukaryotic initiation factor 4A-I (EIF4A1), aspartate aminotransferase (GOT1), and ubiquitin (Ub) (Fig 4B and S2 Fig). In addition to TDP-43, we also analyzed two additional model proteins that use different mechanisms to form pathological aggregates. HTTQ103 consists of a stretch of 103 polyglutamine residues deriving from the abnormal CAG expansion found in Huntington disease–causing huntingtin mutant [21]. GA50 (50 Glycine-Alanine repeats) is derived from the abnormal GGGGCC repeats found in the ALS-causing C9orf72 intron [22]. Both at the microscopic level and by the filter trap assay, aggregates of HTTQ103 and GA50 were also markedly reduced by Hero9 (HTTQ103), Hero45 (GA50), and several other Hero proteins (Fig 4A and 4B and S2 Fig). Of note, prevention of pathogenic polyglutamine and polyalanine aggregations in the T-REx293 cell line was previously shown for LEA proteins derived from an anhydrobiotic nematode or soya bean [23,24]. Thus, anti-aggregation may be a common feature for heat-soluble proteins. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Hero proteins prevent pathogenic protein aggregation in cells. (A) Representative microscopic images of GFP signals in the cells transfected with GFP-tagged TDP-43ΔNLS, HTTQ103, or GA50, together with the GST control or each Hero protein. Note that signals from aggregations are saturated. (B) Filter trap assay of aggregation-prone proteins expressed in cells. TDP-43ΔNLS, HTTQ103, or GA50 was expressed in a GFP-fusion form in HEK293T cells, together with each Hero protein or the GST control. The original or 5-fold diluted samples were loaded on a cellulose acetate membrane in the presence of 1% SDS and the trapped aggregates were probed with anti-GFP antibody. (C) Filter trap assay of aggregation-prone proteins expressed in cells. GFP-TDP-43ΔNLS was expressed in HEK293T cells, together with each Hero protein, its KR-less mutant, in which positively charged amino acids (Lys and Arg) were substituted with neutral Gly, or the GST negative control. Five-fold serial dilutions of the cell lysates were subjected to the filter trap assay and probed with the anti-GFP antibody. Experiments were simultaneously performed, and the presented data were spliced from the same membrane. (D) Filter trap assay of aggregation-prone proteins expressed in cells. GFP-TDP-43ΔNLS was expressed in HEK293T cells, together with the GST control, each Hero protein, or their shuffled amino acid sequences with 42– or 100–amino acid length. Five-fold serial dilutions of the cell lysates were subjected to the filter trap assay and probed with anti-GFP antibody. Experiments were simultaneously performed, and the presented data were spliced from the same membrane. aa, amino acid; GFP, green fluorescent protein; GST, glutathione S-transferase; Hero, heat-resistant obscure; KR, lysine and arginine; TDP-43ΔNLS, TDP-43 lacking the nuclear localization signal; WT, wild-type. https://doi.org/10.1371/journal.pbio.3000632.g004 Interestingly, among the six Hero proteins tested, there was no “super-Hero” protein able to function for all proteins. Instead, distinct subsets of human Hero proteins were more effective in preventing different types of aggregations (Fig 4B). For example, Hero11 promoted rather than prevented aggregation of GA50 compared to GST, while strongly suppressing that of TDP-43ΔNLS and HTTQ103 (Fig 4B). Similarly, Hero7 was previously shown to promote aggregation of HA-tagged HTTQ74 in SK-N-SH cells [25], whereas we found that it strongly suppressed aggregation of GFP-tagged TDP-43ΔNLS in HEK293T cells (Fig 4B). We suggest that different Hero proteins have different preferences for their protein “clients,” which may vary depending on the cellular environments. Nevertheless, it is remarkable that, for a variety of assays with different client proteins and experimental settings (Figs 3 and 4), there were always multiple Hero proteins (despite looking at only six out of hundreds of putative Hero proteins in humans) that showed strong protein-stabilization and anti-aggregation effects.

Extreme charges of Hero proteins are necessary and sufficient to suppress TDP-43 aggregations Apparently, there was no common rule for the effective combination between different Hero proteins and different client proteins. However, we noticed that Hero45, -7, and -11, which were particularly effective in preventing the cellular aggregation of TDP-43ΔNLS (Fig 4B), have unusually high positive charges (pI = 8.66, 10.44, and 11.55, respectively), with high proportions of basic amino acid residues (proportion of Arg + Lys = 18.4%, 32.2%, and 27.3%, respectively). This raised a possibility that these positively charged properties may be important for their functions to prevent TDP-43ΔNLS aggregation. To confirm this idea, we constructed mutants of Hero7 and Hero11, in which Lys and Arg (positively charged) were replaced to Gly (non-charged). As shown in Fig 4C, lysine and arginine (KR)-less mutants of Hero7 and Hero11 have largely lost the anti-aggregation activity, suggesting that positive charges of Hero proteins are necessary for this activity, at least for TDP-43 aggregation. To test the sufficiency of positive charges of Hero proteins, we next randomly scrambled the amino acid sequences of Hero7 and -11, and GST as a control, while keeping their original amino acid composition ratio (i.e., percentage of each amino acid) and fixing the total amino acid lengths at 100 amino acids (aa) or 42-aa (Fig 4D and S3 Table; three different shuffles for 100-aa and two different shuffles for 42-aa). Strikingly, the randomized 100-aa sequences, derived from extremely positively charged Hero7 and -11, prevented the aggregation of TDP-43ΔNLS with comparable or even higher efficiencies than the original ones. Importantly, none of the scrambled 42-aa sequences were able to prevent the TDP-43ΔNLS aggregation, regardless of their amino acid compositions. These results indicate that their amino acid composition and length (i.e., their molecular nature as long, hydrophilic, and highly charged “polymers”), but not their amino acid sequence per se, are the key for Hero proteins to impart their client-protecting functions, at least for TDP-43ΔNLS. Of note, it was previously shown that short peptide fragments of randomly chosen IDPs in humans (35–45 amino acids) have weak cryoprotective and lyophilization-protective activities (up to 1.3–1.5-fold compared to BSA) [26], suggesting that the amino acid length is also important for protecting other proteins under stress conditions.

Hero proteins suppress the neurotoxicity caused by protein aggregates in motor neurons We next examined the anti-aggregation activity of Hero proteins in human induced-pluripotent stem (iPS)-derived motor neurons. We expressed GFP-tagged TDP-43ΔNLS together with each of the six representative human Hero proteins or the GST negative control, as in HEK293T cells. Aggregates of GFP-tagged TDP-43ΔNLS gave saturated fluorescent signals, which were suppressed by co-expression of Hero7 (Fig 5A). To quantify the degree of aggregation, we defined the ratio of the saturated area as the aggregation index (Fig 5B). Similarly to the analysis in HEK293T cells (Fig 4B), Hero9, -45, -7, and -11 strongly reduced the aggregation formation in motor neurons (Fig 5C). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Hero proteins suppress the neurotoxicity of TDP-43ΔNLS aggregates in motor neurons. (A) Representative images of aggregation in human iPS-derived motor neurons transfected with GFP-TDP-43ΔNLS, together with the GST negative control or Hero7. Note that signals from aggregations are saturated. (B) Scheme for counting the saturated area of TDP-43ΔNLS aggregates. (C) Aggregation index analysis in motor neurons. GFP-TDP-43ΔNLS was expressed in motor neurons, together with each Hero protein or the GST negative control. Data indicate normalized saturated area (aggregation index), and represent means ± SD from 3 independent experiments. Hero9, -45, -7, and -11 reduced the aggregate formation. (D) Neurite length analysis in motor neurons. Each Hero protein or the GST control was individually expressed in motor neurons. Data indicate neurite length, and represent means ± SD from 8 cells. Hero45 showed a modest defect in neurite outgrowth. (E) Representative images of the neuron marker TUJ1 in motor neurons transfected with GFP-TDP-43ΔNLS, together with Hero7 or the GST control. Note that signals are saturated to clearly visualize the neurites. (F) Neurite length quantification in motor neurons transfected with GFP-TDP-43ΔNLS together with each Hero protein or the GST negative control. Data indicate neurite length, and represent means ± SD from 8 cells. Hero9, -7 and -11 rescued the neurite outgrowth defect by GFP-TDP-43ΔNLS aggregation. The numerical data pertaining to this figure can be found in S1 Data file. GFP, green fluorescence protein; GST, glutathione S-transferase; Hero, heat-resistant obscure; iPS, induced-pluripotent stem; TDP-43ΔNLS, TDP-43 lacking the nuclear localization signal; TUJ1, neuron-specific class III beta-tublin. https://doi.org/10.1371/journal.pbio.3000632.g005 The neurite length is widely used as a measurement of the neuronal condition. Indeed, it is known that ALS patient–derived motor neurons with mutations in TDP-43 have shorter neurites compared to those from healthy donors [27]. To evaluate neuronal protective activity of Hero proteins, we measured the total neurite length of the transfected neurons. Except for Hero45, overexpression of Hero proteins themselves had no or little effect on neuronal morphology (Fig 5D). In contrast, overexpression of GFP-TDP-43ΔNLS markedly reduced the neurite outgrowth to approximately 30% compared to that of control neurons without aggregation (Fig 5F). However, co-expression of Hero9, -7, and -11 almost fully rescued the neurite outgrowth defects by TDP-43 aggregation (Fig 5E and 5F). Therefore, Hero proteins can efficiently protect human motor neurons from the neurotoxicity of TDP43 aggregation.

Hero proteins suppress the neurotoxicity of protein aggregates in Drosophila eyes Hero proteins’ protective activity can be observed not only in cultured cells but also in Drosophila in vivo. The fly eye has been used as a valuable model system for studying neurodegenerative diseases. Indeed, expression of human TDP-43 fused with yellow fluorescent protein (YFP) (TDP-43-YFP) in the differentiating photoreceptor cells using a glass multimer reporter (GMR)-Gal4 driver causes retinal degeneration [28] (Fig 6A, middle). We generated transgenic flies expressing human Hero9, -13, -45 or -11 in the retina and introduced the transgenes into strains expressing TDP-43-YFP. Strikingly, the eye degeneration by TDP-43-YFP was almost completely suppressed by co-expression of Hero9 (Fig 6A, middle). The rescue effect of TDP-43-YFP by Hero45 was moderate but could be boosted by doubling the copy number of the transgene (Fig 6A, bottom). There were no harmful effects by retina-specific expression of Hero proteins per se; neither YFP nor any of the transgenic Hero proteins alone caused a detectable change in the normal eye morphology (Fig 6A, top). These data show that Hero proteins can suppress pathogenic protein aggregations in the living fly eye. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Hero proteins suppress the neurotoxicity of protein aggregates in Drosophila eyes. (A) Expression of Hero9, -13, -45, or -11 alone showed no phenotype in the external eye morphology, much as the YFP control (upper). Expression of TDP-43-YFP caused the external eye degeneration. Hero9 and double copies of Hero45 strongly suppressed the eye degeneration by TDP-43-YFP (middle and lower). (B) Knockdown of Hsc70-4 or CG17931 caused detectable external eye degeneration, compared to the mock knockdown of Piwi. (C) Nail polish imprint of eyes. Knockdown of Hsc70-4 or CG17931 caused irregular arrangements of ommatidia and bristles. (D) Knockdown of Hsc70-4 or CG17931 decreased the retinal depth. Arrowhead in the left picture indicates the retinal depth. Data represent means ± SD from 5 individual flies. (E) Knockdown Hsc70-4, CG17931, CG14818, or Vig2 caused exacerbation of the eye morphology defect by overexpression of TDP-43-YFP (upper). Knockdown of fly Hero proteins, CG17931, CG12384, or CG11444 caused exacerbation of the eye morphology defect by overexpression of aggregation-prone MJDtr-Q78 (lower). Note that female flies were used for (E), while male flies were used for (A–D). We observed the same phenotype for at least 10 flies per each genotype in each assay. The numerical data pertaining to this figure can be found in S1 Data file. Hero, heat-resistant obscure; Hsc, heat shock cognate; MJDtr-Q78, Machado-Joseph disease truncated-poly-glutamine 78; RNAi, RNA interference; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3000632.g006 Using this Drosophila eye system, we next investigated functions of the endogenous Hero proteins. It is known that Hsc70-4, the most abundant and constitutively expressed Hsp70 chaperone, is required for normal eye development in Drosophila [29–31]. Indeed, knockdown of Hsc70-4 in the eyes showed a modest but detectable defect in external eye morphology, whereas mock depletion of germline-specific Piwi caused no abnormality (Fig 6B). Interestingly, eye-specific knockdown of CG17931, a homolog of human Hero7 (identity = 58%, similarity = 68%), also showed a similar eye degeneration phenotype as that of Hsc70-4. Moreover, irregular arrangements of ommatidia and bristles (Fig 6C), as well as reduction of the retinal depth (Fig 6D), were also caused by knockdown of Hsc70-4 or CG17931. These observations suggest that not only chaperones but Hero proteins also play important roles in fly eye development. We then evaluated the protective effect of endogenous fly Hero proteins against pathogenic protein aggregates. To this end, we utilized the abovementioned aggregation-prone TDP-43-YFP (Fig 6E, upper) as well as human Ataxin3 containing a pathogenic expansion of 78 glutamine repeats (Machado-Joseph disease truncated-polyglutamine 78 [MJDtr-Q78]; Fig 6E, bottom), which also causes retinal degeneration when overexpressed in Drosophila eyes [32] (Fig 6E, bottom). Strikingly, defects by these aggregation-prone proteins were markedly exacerbated by simultaneous knockdown of the Hero7 homolog CG17931, compared to the negative control of Piwi knockdown (Fig 6E). Exacerbated eye phenotypes with MJDtr-Q78 and TDP-43-YFP were also apparent in the eye depleted of other endogenous fly Hero proteins, such as CG14818 and Vig2, which are potential homologs of human Hero9 (identity = 23%, similarity = 40%) and Hero45 (identity = 30%, similarity = 41%), respectively, and CG12384 and CG11444 (Fig 6E). Together, these data show that Hero proteins act to suppress aggregation-associated eye degeneration in living flies, consistent with our in cell data (Figs 4 and 5).

Hero proteins are essential both in cells and in vivo To understand Hero proteins’ functions in non-overexpressed, physiological conditions, we performed clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockout (KO) of six Hero proteins in HEK293T cells. We found that KO of Hero13 and 7 showed defects in cell proliferation under the normal culture condition (Fig 7A). Moreover, whole-body knockdown of endogenous Hero proteins in Drosophila using actin-GAL4-driven long hairpin RNAs often caused lethality (Fig 7B). Thus, Hero proteins play essential roles in human cell proliferation and Drosophila early development. We note that Frost in Drosophila, which is highly up-regulated after cold shock [33] and required for maintaining female fertility following cold exposure [34], is extremely disordered and charged, and presumably represents another example of Hero proteins with physiological functions in flies. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Physiological functions of Hero proteins. (A) Cell growth assay of HEK293T cells acutely knocked out for the indicated genes. Four days after transfection of gRNA, the numbers of the cells were measured by a coloring assay. Knockout of Hero13 and Hero7 reduced the cell proliferation. Data represent means ± SD of the cell numbers from 8 independent wells. (B) Whole-body knockdown phenotypes in Drosophila. Knockdown of endogenous fly Hero proteins were performed by two independent RNAi strains (GD and KK lines). Whole-body depletion of Hero proteins often caused lethality, suggesting that Hero proteins play essential roles in Drosophila early development. Note that we used two different GD lines for CG3760. We observed the same phenotype for at least two independent crosses per each genotype. (C) Survival plots of Drosophila. Whole-body overexpression of CG12384 or CG14818 elongated life spans, compared to the wild type. (D) Summary of life span analysis in Drosophila. Whole-body overexpression of Hero proteins except human Hero11 significantly elongated life spans. (E) A model for the modes of action of chaperones and Hero proteins. Chaperones usually act on the inactive/denatured state of their client proteins and actively reverse them to the functional state by using ATP, while Hero proteins likely act on the functional state of their client proteins and protect them from lapsing into the inactive/denatured state in an ATP-independent manner. The numerical data pertaining to this figure can be found in S1 Data file. gRNA, guide RNA; Hero, heat-resistant obscure; RNAi, RNA interference; WT, wild-type; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3000632.g007