Article |
Address correspondence to M.Y. Sherman, Boston University School of Medicine, Dept. of Biochemistry, K323, 715 Albany St., Boston, MA 02118. Tel.: (617) 638-5971. Fax: (617) 638-5339. E-mail: sherman{at}biochem.bumc.bu.edu
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Abstract |
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Key Words: aggregation; polyglutamine; toxicity; prions; yeast
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Introduction |
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It was initially assumed that denatured or abnormal proteins aggregate and form IBs simply due to their intrinsic tendency to associate with each other until the aggregates eventually become insoluble. However, recent reports demonstrated that formation of cytoplasmic IBs in mammalian cells may be a complex process involving transport of small protein aggregates from the cell's periphery to the centrosome along microtubules. These IBs contain Hsps, ubiquitin, ubiquitin-conjugating enzymes, and 26S proteasomes forming a large structure termed aggresome (Vidair et al., 1996; Wojcik et al., 1996; Johnston et al., 1998; Anton et al., 1999; Garcia-Mata et al., 1999; Wigley et al., 1999; Fabunmi et al., 2000). Recent findings from our and other groups indicate that IBs formed by polyQ-containing polypeptides have similarities with the aggresome (Stenoien et al., 1999; Wyttenbach et al., 2000; Meriin et al., 2001; Waelter et al., 2001). Furthermore, we have found recently that a stress-activated protein kinase MEKK1 stimulates early stages of IB formation (Meriin et al., 2001), suggesting that various steps of IB formation are tightly controlled by the cell.
Close correlation between formation of the huntingtin-containing IBs and death of the affected neurons suggests that IB may be involved in neurodegeneration (Davies et al., 1998). However, there is evidence that IB formation may not be necessary for the neuronal death, since artificial inhibition of aggregation of polyQ-containing polypeptides in certain animal and cellular models did not inhibit and even enhanced neuronal apoptosis (Klement et al., 1998; Saudou et al., 1998; Cummings et al., 1999b). Therefore, IB formation may be irrelevant to the neurodegeneration process or may even serve a protective role by capturing toxic soluble polyQ molecules (for review see Sherman and Goldberg, 2001). The question whether soluble abnormal proteins or IBs cause toxicity and neurodegeneration is the focus of ongoing discussion in the field. Because of the complexity of polyQ-induced neuronal death and IB formation, development of adequate cellular and animal models is critical to dissect cellular mechanisms of these processes. The practical application of this research would be identification of proteins involved in neurodegeneration and IB formation in order to use them as targets for drug design.
In mice, Drosophila and Caenorhabditis elegans expression of extended polyQ polypeptides caused toxicity and neurodegeneration, and IBs in neurons were detectable, whereas polypeptides with polyQ of normal length were not toxic and did not form IBs (Davies et al., 1997; Scherzinger et al., 1997; Kazemi-Esfarjani and Benzer, 2000; Satyal et al., 2000). Notably, in mice model expression of exon 1 of huntingtin, a small NH2-terminal fragment with extended polyQ domain, was sufficient to cause both neurodegeneration and IB formation (Davies et al., 1997). Yeast Saccharomyces cerevisiae has also been used as a model, since it provides a useful tool for screening of genes involved in IB formation and potential polyQ-induced toxicity and for screening of chemical compounds, which inhibit these processes. Short polyQ was shown to be soluble in yeast, whereas long polyQ polypeptideformed IBs; however, no toxicity of polyQ polypeptides in yeast has been reported so far (Krobitsch and Lindquist, 2000; Muchowski et al., 2000). In these yeast models, the molecular chaperone Hsp104 was reported to be essential for the aggregation of polyQ (Krobitsch and Lindquist, 2000). Overexpression of other molecular chaperones Hsp70 and Hdj1 also affected polyQ aggregation suppressing formation of fibrous aggregates and promoting instead formation of amorphous structures (Muchowski et al., 2000).
The molecular chaperones Hsp104, Hsp70, and Sis1 were implicated in emergence and maintenance of prion conformation of certain yeast proteins, for example, Sup35, Rnq1, Ure2 (Chernoff et al., 1995, 1999; Derkatch et al., 1997; Newnam et al., 1999; Jung et al., 2000; Moriyama et al., 2000; Sondheimer and Lindquist, 2000; Sondheimer et al., 2001; Wegrzyn et al., 2001). These polypeptides in prion conformation aggregate and convert normal polypeptides of the same type into prion conformation, thus recruiting them into IBs. The phenotypic traits resulting from such aggregation are inherited in a non-Mendelian fashion (Wickner et al., 1999; Serio and Lindquist, 2000). These mechanisms closely resemble aggregation of mammalian prion PrP, a cause of a group of neurodegenerative disorders (Prusiner, 2001).
Here, we develop a new yeast model of polyQ expansion diseases, which establishes a direct link between polyQ aggregation and toxicity. Using this model we searched for cellular elements involved in control of polyQ aggregation and toxicity.
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Results |
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To study whether expression of expanded polyQ inhibits growth by affecting the cell cycle progression, we compared cellular DNA contents in the cultures expressing 25Q and 103Q. Cells induced in selective galactose medium for 20 h were stained with propidium iodide and then analyzed by FACS®. Expression of 103Q in strain W303 caused a slight increase of G2 phase cells in comparison with cells expressing 25Q (unpublished data), whereas in 103Q-expressing JN54 cells no changes in the cell cycle were observed (Fig. 1 C) in spite of significantly decreased growth. This indicates that there is no substantial cell cycle delay, which could account for the growth inhibition upon accumulation of 103Q.
Molecular chaperones are essential for polyQ aggregation and toxicity
Microscopic observation showed that 25Q was diffusely distributed in yeast cells, whereas every cell expressing 103Q formed multiple aggregates of two types (Fig. 2 A): appearing either as large disordered lumps or as small grains (or flakes), some of which were swarming in a cell. A fraction of cells with small aggregates increased in expense of cells with large aggregates upon prolonged 103Q accumulation, suggesting that aggregates are dynamic formations capable of further evolution. Fractionation of cell homogenates in the presence of 1% Triton X-100 provided independent evidence that the majority of 25Q (>75%) were soluble in the cells (Fig. 2 B). Notably, a fraction of the 25Q was found in 800 g pellets, indicating that although visible aggregates were not seen with short polyQ, some of the molecules associated with large detergent-insoluble cellular structures. With 103Q, from 50 up to 95% of the polypeptide was found in pellets, depending on duration of induction and yeast strain.
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The differences in 103Q aggregation patterns in the chaperone mutants may reflect different roles of the chaperones in polyQ aggregation. We followed the process of aggregation in wild-type and mutant cells in real time by taking pictures every 2 min of several selected cells expressing 103Q. In the wild-type, initially all fluorescent cells contained small aggregates. With time, intensity of fluorescence in aggregates and aggregate size grew, whereas the soluble (diffusely distributed) fluorescent material gradually disappeared (Fig. 3). However, even after 60 min of observation a certain pool of 103Q still remained soluble. Unlike the wild-type cells, in ssa1 ssa2 cells no apparent redistribution of soluble 103Q into numerous aggregates was seen within 60 min (unpublished data), indicating that although the nucleation of aggregates is unimpeded in this mutant, further growth of the aggregates is inhibited. We monitored rare events of IB formation in the hsp104 mutant and found a remarkable difference with polyQ aggregation compared with the wild-type cells (Fig. 3). Once started, the aggregation of 103Q in hsp104 mutant proceeded rapidly, so 612 min after the first appearance of visible seeds almost all soluble polyQ in the cell collapsed into them, forming large IBs (Fig. 3). Therefore, the rate-limiting step in the hsp104 mutant is not growth of prenucleated aggregates but initiation of the aggregation process. In a small number of cells where aggregates are seeded despite the lack of Hsp104, the consequent growth of the IB proceeds rapidly. Therefore, Hsp104 appears to be involved (possibly indirectly as discussed below) in nucleation of polyQ aggregates, whereas members of the Ssa family appear to be involved in expansion of prenucleated aggregates, which defines the difference in aggregation phenotypes in the chaperone mutants.
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The conclusive proof of such type of inheritance came from sporulation/dissection of diploids resulting from mating of the mutants with the wild-type strain. All four spores in a tetrad usually displayed wild-typelike aggregation and toxicity of 103Q, indicating a non-Mendelian mode of inheritance. In contrast, sporulation and dissection of diploids originating from a mating of wild-type strain and hsp104 mutant resulted in 2:2 segregation of aggregation/toxicity pattern. In these tetrads, suppressed aggregation was always associated with the hsp104 deletion. The non-Mendelian mode of inheritance of polyQ and toxicity was reminiscent of inheritance of prion elements in yeast (Wickner et al., 1999; Sondheimer and Lindquist, 2000). Since both Hsp104 and Sis1 have been implicated in prion propagation (Chernoff et al., 1995; Derkatch et al., 1997; Moriyama et al., 2000; Sondheimer and Lindquist, 2000; Sondheimer et al., 2001), we suggested that some protein(s) in prion conformation may be essential for toxicity and aggregation of polyQ polypeptides.
To test this possibility, we screened a set of deletion mutations in nonessential prion genes and in genes involved in aggregation of prion proteins (i.e., mks1, new1, sla1, rnq1, ure2, and ybr016w) for their effects on polyQ aggregation. Of all of the mutants tested, only cells with the rnq1 deletion displayed an aggregation defect. The 103Q aggregation pattern in these cells was similar to that of hsp104 and sis185 mutants (Fig. 5 A, middle). No 103Q-related toxicity was detected in rnq1 mutant cells (Fig. 5 B) despite the fact that 103Q was accumulated to higher levels than in the wild-type strain (Fig. 5 C). Mating of the rnq1 mutant with the isogenic wild-type strain followed by sporulation and tetrad dissection resulted in a 2:2 segregation of suppressed aggregation to toxicity as was shown before with the hsp104 mutant. Suppressed aggregation was always associated with rnq1 deletion. These data strongly indicate that Rnq1 is essential for aggregation and toxicity of polyQ polypeptides in yeast. Using a differential centrifugation of cell homogenates, we found that in W303 and JN54, the wild-type strains used in this study, all Rnq1 protein was associated with 100,000 g pellets, indicating that this protein exists in prion form (Fig. 5 D).
To investigate whether prion conformation of Rnq1 is critical for polyQ aggregation, we employed a method of curing yeast cells of prions by growing them in the presence of guanidine hydrochloride (GuHCl), which prevents propagation of prion conformation (for review see Chernoff, 2001). Accordingly, yeast cells were grown for three passages on glucose-containing medium (to block 103Q induction) in the presence of 5 mM GuHCl. Expression of 103Q was induced in 22 randomly chosen individual clones originating from this procedure, and 21 of them displayed the aggregation phenotype similar to that of the spontaneous mutants described above (Fig. 5 A, bottom). Moreover, none of the 21 clones demonstrated a growth defect in response to accumulation of 103Q (Fig. 5 B) despite 103Q levels being significantly higher than the levels in the original cells (Fig. 5 C), which underscores the importance of polyQ aggregation for its toxicity. In two randomly chosen clones with no polyQ aggregation, all Rnq1 was found in the 100,000 g supernatant fraction in contrast to the original strain where most of Rnq1 was found in the insoluble fraction (Fig. 5 D). This confirms that the GuHCl treatment indeed cured cells of prion form of Rnq1 ([RNQ+]). These data strongly suggest that prion conformation of Rnq1 is essential for aggregation and toxicity of polyQ.
Since hsp104 and sis1 mutations are known to cure cells of [RNQ+] (Derkatch et al., 1997; Sondheimer et al., 2001), it appears that suppression of polyQ aggregation and toxicity by these mutations is caused primarily by the loss of Rnq1 prion. On the other hand, the ssa1ssa2 and ydj1151 mutations, which also relieved polyQ-related toxicity (Fig. 4), did not affect prion state of Rnq1, since all Rnq1 remain associated with the insoluble fraction in the ssa1ssa2 or ydj1151 cells homogenates (Fig. 5 D). Therefore, effects of ssa1ssa2 and ydj1151 mutations appear to be unrelated to [RNQ+]. These data support the notion that Ssa and Ydj1 apparently affect a step in IB formation different from one controlled by Hsp104 and Sis1.
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Discussion |
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Our data indicate that in addition to genetic factors the presence of prions determines polyQ aggregation and consequently cytotoxicity in yeast cells. It is possible that the lack of polyQ toxicity in previously reported yeast models may be related to the lack of [RNQ+] prion in the strains used in those experiments (Krobitsch and Lindquist, 2000; Hughes et al., 2001). This suggestion is supported by the morphology of aggregates in these reports, which resembled that in our clones cured of [RNQ+] prion. Another critical factor in toxicity appears to be the intracellular level of expanded polyQ (unpublished data).
Our data demonstrate that, counterintuitively and in contrast to in vitro results, aggregation of polyQ in vivo is strongly dependent on cellular factors. We found that among chaperones, in addition to Hsp104 (Krobitsch and Lindquist, 2000), Sis1, Ssa1/2, and Ydj1 also play an essential role in formation of the IBs. The majority of cells mutated in either hsp104 or sis1 genes were unable to form any visible 103Q aggregates. In rare hsp104 mutant cells in which nucleation of aggregates occurred, IBs grew efficiently. These data suggest that Hsp104 (and probably Sis1) is not essential for expansion of aggregates but rather for seeding of IBs. Likely, these chaperones control seeding of aggregates via maintenance of the [RNQ+] prion (Derkatch et al., 1997; Sondheimer et al., 2001; Wegrzyn et al., 2001). In addition, Hsp104 could be directly involved in shearing of polyQ aggregates. Such a role for Hsp104 would be consistent with its involvement in shearing of Sup35 aggregates (Patino et al., 1996; Paushkin et al., 1996), which is critical for maintenance of prion form of Sup35 ([PSI+]) (for review see Chernoff, 2001; Wegrzyn et al., 2001).
Ssa1/Ssa2 and Ydj1 were also essential for formation of IBs but at a different step. Real-time observation of this process indicated that Ssa1/Ssa2 (possibly with the help of Ydj1) might be involved in expansion of preformed seeds. This agrees with the previously proposed role of Ssa in the growth of prion aggregates in yeast (for review see Chernoff, 2001). In cells with decreased levels of Ssa (or deprived of Ydj1), growth of aggregates might become a limiting step, and some 103Q molecules unable to join IBs would remain soluble, which in turn would allow more nucleation centers to emerge in a cell and a much higher number of small aggregates to be formed. Together these data indicate a novel and unexpected role of molecular chaperones in promoting formation of protein aggregates. Furthermore, different chaperones facilitate two distinct steps in the aggregation process. Interestingly, although physiological levels of chaperones are necessary for the aggregation, overexpression of Hsp104, Hsp70, and Hsp40 was reported to keep polyQ polypeptides in a soluble form (Chan et al., 2000; Satyal et al., 2000). However, it should be noted that it is not clear whether strains used in these studies contained the [RNQ+] prion.
Our data that the polypeptide with expanded polyQ forms inclusions only in the presence of the [RNQ+] prion are consistent with the latest report showing that aggregation of the MJD protein with polyQ expansion is facilitated in so-called [PIN+] cells. [PIN+] is a non-Mendelian genetic trait promoting emergence of [PSI+] (Derkatch et al., 1997). It was shown that [PIN+] may be caused by a number of potential prion-forming proteins, for example, Rnq1 and New1 (Derkatch et al., 2001). Interestingly, deletion of the NEW1 gene did not significantly affect aggregation of 103Q polypeptide, suggesting the major role of [RNQ+] prion in this process. Whether aggregates of prion form of Rnq1, which has a QN-rich domain, could directly serve as a nucleation site for polyQ, or Rnq1 acts on the polyQ aggregation indirectly is yet to be established.
Our data show that the toxicity of expanded polyQ polypeptide in yeast cells depends on the presence of prion in a cell. This fact suggests that in mammalian tissues some (possibly other than PrP) prion-like proteins may be required for aggregation and neuronal pathology of mutant polypeptides with expansion of the polyglutamine tract. This consideration establishes a link between the two seemingly distant groups of neurodegenerative diseases.
Presented here is a yeast model of cytotoxicity caused by polyQ aggregation, which can be further used in a search for yeast genetic modifiers of aggregation and viability. The identification of such cellular factors will ultimately facilitate the quest for genetic modifiers in humans and be crucial for understanding the mechanisms of the development of polyQ expansion-related pathology.
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Materials and methods |
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Observation of yeast growth and induction
Cells were routinely grown at 30°C on selective SD medium and then transferred into SG liquid or solid medium for induction of 25Q or 103Q expression.
To stain cells with propidium iodide for flow cytometry, 0.55 ml of cells grown in SG medium overnight were harvested, washed with 50 mM Tris-HCl (pH 7.5), and treated as follows. Cells were fixed in 70% ethanol for 1 h, washed, and treated with 1 mg/ml RNase A in 50 mM Tris-HCl (pH 7.5) for 1 h with rotation. Then, cells were treated with 0.5% pepsin in 55 mM HCl for 5 min and incubated with 0.05 mg/ml propidium iodide in 180 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 7 mM MgCl2 overnight at 4°C with rotation.
Microscopy
Confocal microscopy was performed with laser scanning system Radiance 2000 (Bio-Rad Laboratories). To follow a process of aggregation in real time, 103Q was induced for 3 h, cells were harvested, mounted on glass slides covered with solid (1.5% agarose) galactose medium, and observed under the deconvolution epifluorescent microscope (Deltavision; Applied Precision, Inc.). Every 2 min, 1015 serial sections (0.25 mm/section, 0.5 s exposure/section) were taken to cover the whole thickness of the cell for a total of 1 h.
Analysis of solubility of polyQ polypeptides and Rnq1 protein in cell lysates
Collected cells were disrupted by vortexing with 425600 µm acid-washed glass beads in 40 mM Hepes, pH 7.5, 50 mM KCl, 1% Triton X-100, 2 mM DTT, 1 mM Na3VO4, 50 mM ß-glycerophosphate, 50 mM NaF, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 1 mM benzamidine, and 5 µg/ml each of leupeptin, pepstatin A, and aprotinin. Disintegration by lyticase treatment provided similar results (unpublished data). After disintegration, cell lysates were left in narrow tubes for 1 h to allow 95% of unbroken cells to sediment by the gravity force, whereas almost all of the aggregates released from the broken cells stayed in the solution (unpublished data). The upper portion of supernatant was carefully removed and used for fractionation. Samples were normalized by the amount of total protein. Lysates were subjected to centrifugation at 800 g for 10 min. The pellets were washed once with the lysis buffer and resuspended in a volume of water equal to the volume of the supernatant. To assess intracellular aggregation of Rnq1, cells were disrupted by vortexing with glass beads in the buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM KCl, 100 mM EDTA, 1 mM DTT, 1.0% Triton X-100, and 0.2% SDS. Then cell homogenate was cleared from debris by 10 min centrifugation at 3,000 g and subjected to centrifugation at 100,000 g to separate soluble and insoluble forms of the protein. All samples were supplemented with loading SDS-PAGE buffer containing 2% SDS and boiled for 3 min before being subjected to immunoblotting. GFP-tagged polyQ polypeptides were visualized with anti-GFP antibody (polyclonal) (CLONTECH Laboratories, Inc.). Rnq1 antibody was a gift from S. Lindquist (Whitehead Institute, Cambridge, MA).
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Footnotes |
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Acknowledgments |
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Submitted: 20 December 2001
Revised: 1 April 2002
Accepted: 17 April 2002
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References |
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