Hsp104 Overexpression Research Articles

Hsp70/Hsp90 co‐chaperones are required for efficient Hsp104‐mediated elimination of the yeast [PSI+] prion but not for prion propagation

The continued propagation of the yeast [PSI(+)] prion requires the molecular chaperone Hsp104 yet in cells engineered to overexpress Hsp104; prion propagation is impaired leading to the rapid appearance of prion-free [psi(-)] cells. The underlying mechanism of prion loss in such cells is unknown but is assumed to be due to the complete dissolution of the prion aggregates by the ATP-dependent disaggregase activity of this chaperone. To further explore the mechanism, we have sought to identify cellular factors required for prion loss in such cells. Sti1p and Cpr7p are co-chaperones that modulate the activity of Hsp70/Ssa and Hsp90 chaperones and bind to the C-terminus of Hsp104. Neither Sti1p nor Cpr7p is necessary for prion propagation but we show that deletion of the STI1 and CPR7 genes leads to a significant reduction in the generation of [psi(-)] cells by Hsp104 overexpression. Deletion of the STI1 and CPR7 genes does not modify the elimination of [PSI(+)] by guanidine hydrochloride, which inhibits the ATPase activity of Hsp104 but does block elimination of [PSI(+)] by overexpression of either an ATPase-defective mutant of Hsp104 (hsp104(K218T/K620T)) or a 'trap' mutant Hsp104 (hsp104(E285Q/E687Q)) that can bind its substrate but can not release it. These results provide support for the hypothesis that [PSI(+)] elimination by Hsp104 overexpression is not simply a consequence of complete dissolution of the prion aggregates but rather is through a mechanism distinct from the remodelling activity of Hsp104.

A prion of yeast metacaspase homolog (Mca1p) detected by a genetic screen

Saccharomyces cerevisiae can be infected with four amyloid-based prions: [URE3], [PSI(+)], [PIN(+)], and [SWI(+)], due to self-propagating aggregation of Ure2p, Sup35p, Rnq1p and Swi1p, respectively. We searched for new prions of yeast by fusing random segments of yeast DNA to SUP35MC, encoding the Sup35 protein lacking its own prion domain, selecting clones in which Sup35MC function was impaired. Three different clones contained parts of the Q/N-rich amino-terminal domain of Mca1p/Yca1p with the Sup35 part of the fusion protein partially inactive. This inactivity was dominant, segregated 4:0 in meiosis, and was efficiently transferred by cytoplasmic mixing. The inactivity was cured by overexpression of Hsp104, but the prion could arise again in the cured strain (reversible curing). Overproduction of the Mca1 N-terminal domain induced the de novo appearance of the prion form of the fusion. The prion state, which we name [MCA], was transmitted to the chromosomally encoded Mca1p based on genetic, cytological and biochemical tests.

A G-protein γ subunit mimic is a general antagonist of prion propagation in Saccharomyces cerevisiae

The Gpg1 protein is a Ggamma subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI(+)], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI(+)] but also [PIN(+)], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI(+)] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted alpha-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Delta and gpb1Delta strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.

Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.

Neuroprotection by Hsp104 and Hsp27 in Lentiviral-based Rat Models of Huntington's Disease

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of glutamine repeats in the huntingtin (htt) protein. Abnormal protein folding and the accumulation of mutated htt are hallmarks of HD neuropathology. Heat-shock proteins (hsps), which refold denatured proteins, might therefore mitigate HD. We show here that hsp104 and hsp27 rescue striatal dysfunction in primary neuronal cultures and HD rat models based on lentiviral-mediated overexpression of a mutated htt fragment. In primary rat striatal cultures, production of hsp104 or hsp27 with htt171-82Q restored neuronal nuclei (NeuN)-positive cell density to that measured after infection with vector expressing the wild-type htt fragment (htt171-19Q). In vivo, both chaperones significantly reduced mutated-htt-related loss of DARPP-32 expression. Furthermore, hsps affected the distribution and size of htt inclusions, with the density of neuritic aggregates being remarkably increased in striatal neurons overexpressing hsps. We also found that htt171-82Q induced the up-regulation of endogenous hsp70 that was co-localized with htt inclusions, and that the overexpression of hsp104 and hsp27 modified the subcellular localization of hsp70 that became cytoplasmic. Finally, hsp104 induced the production of endogenous hsp27. These data demonstrate the protective effects of chaperones in mammalian models of HD.

Destruction or Potentiation of Different Prions Catalyzed by Similar Hsp104 Remodeling Activities

Yeast prions are protein-based genetic elements that self-perpetuate changes in protein conformation and function. A protein-remodeling factor, Hsp104, controls the inheritance of several yeast prions, including those formed by Sup35 and Ure2. Perplexingly, deletion of Hsp104 eliminates Sup35 and Ure2 prions, whereas overexpression of Hsp104 purges cells of Sup35 prions, but not Ure2 prions. Here, we used pure components to dissect how Hsp104 regulates prion formation, growth, and division. For both Sup35 and Ure2, Hsp104 catalyzes de novo prion nucleation from soluble, native protein. Using a distinct mechanism, Hsp104 fragments both prions to generate new prion assembly surfaces. For Sup35, the fragmentation endpoint is an ensemble of noninfectious, amyloid-like aggregates and soluble protein that cannot replicate conformation. In vivid distinction, the endpoint of Ure2 fragmentation is short prion fibers with enhanced infectivity and self-replicating ability. These advances explain the distinct effects of Hsp104 on the inheritance of the two prions.

N-Terminal Domain of Yeast Hsp104 Chaperone Is Dispensable for Thermotolerance and Prion Propagation but Necessary for Curing Prions by Hsp104 Overexpression

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of [PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported [PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure [PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI(+)] by direct dissolution of prion aggregates. Curing of [PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI(+)] in proportion to protein resolubilization defects, suggesting that [PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.

Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease

Huntington's disease is a devastating neurodegenerative condition associated with the formation of intraneuronal aggregates by mutant huntingtin. Aggregate formation is a property shared by the nine related diseases caused by polyglutamine codon expansion mutations and also by other neurodegenerative conditions like Parkinsons's disease. The roles of aggregates and aggregation in these diseases have been a subject of heated controversy. Here, we have addressed the question in vivo by generating a new transgenic mouse overexpressing the yeast chaperone hsp104, as hsp104 overexpression reduced mutant huntingtin aggregation and toxicity in cell models. Hsp104 has no close mammalian orthologues and does not appear to have effects on mammalian cell death pathways. We crossed hsp104 transgenic mice with mice expressing the first 171 residues of mutant huntingtin. Hsp104 reduced aggregate formation and prolonged the lifespan of the HD mice by 20%. This protection may be mediated at the level of changing the conformation of a putative toxic monomer, reducing oligomerization or aggregation, reducing the levels of oligomeric species or aggregates or combinations of these non-mutually exclusive possibilities.

Prion-dependent switching between respiratory competence and deficiency in the yeast nam9-1 mutant.

Nam9p is a protein of the mitochondrial ribosome. The respiration-deficient Saccharomyces cerevisiae strain MB43-nam9-1 expresses Nam9-1p containing the point mutation S82L. Respiratory deficiency correlates with a decrease in the steady level of some mitochondrially encoded proteins and the complete lack of mitochondrially encoded cytochrome oxidase subunit 2 (Cox2). De novo synthesis of Cox2 in MB43-nam9-1 is unaffected, indicating that newly synthesized Cox2 is rapidly degraded. Respiratory deficiency of MB43-nam9-1 is overcome by transient overexpression of HSP104, by deletion of HSP104, by transient exposure to guanidine hydrochloride, and by expression of the C-terminal portion of Sup35, indicating an involvement of the yeast prion [PSI(+)]. Respiratory deficiency of MB43-nam9-1 can be reinduced by transfer of cytosol from S. cerevisiae that harbors [PSI(+)]. We conclude that nam9-1 causes respiratory deficiency only in combination with the cytosolic prion [PSI(+)], presenting the first example of a synthetic effect between cytosolic [PSI(+)] and a mutant mitochondrial protein.

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

URE3] and [PSI] are Prions of Yeast and Evidence for New Fungal Prions

[URE3] and [PSI] are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon. Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins. Ure2p is partially protease-resistant in [URE3] strains and Sup35p is aggregated specifically in [PSI] strains supporting this idea. Overexpression of Hsp104 cures [PSI], as does the absence of this protein, suggesting that the prion change of Sup35p in [PSI] strains is aggregation. Strains of [PSI], analogous to those described for scrapie, have now been described as well as an in vitro system for [PSI] propagation. Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora.

The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3.

The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.

Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing.

The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast, hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.