Protein misfolding is the molecular basis for several human diseases. How the primary amino acid sequence triggers misfolding and determines the benign or toxic character of the misfolded protein remains largely obscure. Among proteins that misfold, polyglutamine (polyQ) expansion proteins provide an interesting case: Each causes a distinct neurodegenerative disease that selectively affects different neurons. However, all are broadly expressed and most become toxic when the glutamine expansion exceeds Ϸ39 glutamine residues. The disease-causing polyQ expansion proteins differ profoundly in the amino acids flanking the polyQ region. We therefore hypothesized that these flanking sequences influence the specific toxic character of each polyQ expansion protein. Using a yeast model, we find that sequences flanking the polyQ region of human huntingtin exon I can convert a benign protein to a toxic species and vice versa. Further, we observe that flanking sequences can direct polyQ misfolding to at least two morphologically distinct types of polyQ aggregates. Very tight aggregates always are benign, whereas amorphous aggregates can be toxic. We thereby establish a previously undescribed systematic characterization of the influence of flanking amino acid sequences on polyQ toxicity.

Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.

Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington's disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin. Polyglutamine (polyQ) expansions in proteins are the basis for at least nine different neurodegenerative disorders, including Huntington's disease (HD) (Orr and Zoghbi 2007). The proteins carrying polyQ expansions are broadly expressed, but each disease is characterized by the vulnerability of a particular subset of neurons. Interactions between sequences flanking the polyQ expansion and the proteome unique to distinct neurons must determine the specific character of each disease. However, in virtually every case, toxicity ensues when the expansion reaches ∼40 residues. Further, the age of disease onset decreases and the severity of disease progression increases as the length of the polyQ expansion increases. Thus, even though each disease is distinct, there must be common underlying toxic mechanisms related to polyQmediated misfolding.To investigate polyQ toxicity, we used a combination of yeast, PC12, and striatal cell models. We and others have developed yeast models that express N-terminal fragments of huntingtin (htt exonI) (Krobitsch and Lindquist 2000;Muchowski et al. 2000;Meriin et al. 2002; Duennwald et al. 2006a,b). Our yeast model recapitulates major features of neuronal polyQ pathology, including the hallmark feature of increasing toxicity with increasing polyQ length (Duennwald et al. 2006b). Thus, the yeast model presents the opportunity to identify factors that specifically determine polyQ toxicity in a genetically tractable model organism.Numerous cellular pathways, such as transcriptional regulation (Riley and Orr 2006), vesicular transport (Gunawardena and Goldstein 2005), and protein turnover (Bence et al. 2001;Bennett et al. 2007) are impaired by polyQ expansion proteins. It remains unclear, however, which of these cellular defects are initial and specific triggers of polyQ toxicity. Here, we focused on how the well-established polyQ-induced defect ...

amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed.Hsp40 ͉ neurodegenerative disease ͉ Sis1 ͉ Rnq1 ͉ yeast prion

Safely eradicating prions, amyloids and preamyloid oligomers may ameliorate several fatal neurodegenerative disorders. Yet, whether small-molecule drugs can directly antagonize the entire spectrum of distinct amyloid structures or ‘strains’ that underlie distinct disease states is unclear. Here, we investigated this issue using the yeast prion protein Sup35. We have established how epigallocatechin-3-gallate (EGCG) blocks synthetic Sup35 prionogenesis, eliminates preformed Sup35 prions, and disrupts inter- and intra-molecular prion contacts. Unexpectedly, these direct activities were strain selective, altered the repertoire of accessible infectious forms and facilitated emergence of a new prion strain that configured original, EGCG-resistant intermolecular contacts. In vivo, EGCG cured and prevented induction of susceptible but not resistant strains, and elicited switching from susceptible to resistant forms. Importantly, 4,5-bis-(4-methoxyanilino)phthalimide directly antagonized EGCG-resistant prions and synergized with EGCG to eliminate diverse Sup35 prion strains. Thus, synergistic small-molecule combinations that directly eradicate complete strain repertoires likely hold considerable therapeutic potential.

The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide—either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome. They form an important line of defense against misfolded proteins and are part of the cellular quality control system. The heat shock protein (Hsp) family, particularly Hsp70 and Hsp90, plays a major part in this process and it is well-known to regulate protein misfolding in a variety of diseases, including tau levels and toxicity in AD. However, the role of Hsp90 in regulating protein misfolding is not yet fully understood. For example, knockdown of Hsp90 and its co-chaperones in a Caenorhabditis elegans model of Aβ misfolding leads to increased toxicity. On the other hand, the use of Hsp90 inhibitors in AD mouse models reduces Aβ toxicity, and normalizes synaptic function. Stress-inducible phosphoprotein 1 (STI1), an intracellular co-chaperone, mediates the transfer of clients from Hsp70 to Hsp90. Importantly, STI1 has been shown to regulate aggregation of amyloid-like proteins in yeast. In addition to its intracellular function, STI1 can be secreted by diverse cell types, including astrocytes and microglia and function as a neurotrophic ligand by triggering signaling via the cellular prion protein (PrPC). Extracellular STI1 can prevent Aβ toxic signaling by (i) interfering with Aβ binding to PrPC and (ii) triggering pro-survival signaling cascades. Interestingly, decreased levels of STI1 in C. elegans can also increase toxicity in an amyloid model. In this review, we will discuss the role of intracellular and extracellular STI1 and the Hsp70/Hsp90 chaperone network in mechanisms underlying protein misfolding in neurodegenerative diseases, with particular focus on AD.

Several neurodegenerative diseases are associated with the toxicity of misfolded proteins. This toxicity must arise from a combination of the amino acid sequences of the misfolded proteins and their interactions with other factors in their environment. A particularly compelling example of how profoundly these intramolecular and intermolecular factors can modulate the toxicity of a misfolded protein is provided by the polyglutamine (polyQ) diseases. All of these disorders are caused by glutamine expansions in proteins that are broadly expressed, yet the nature of the proteins that harbor the glutamine expansions and the particular pathologies they produce are very different. We find, using a yeast model, that amino acid sequences that modulate polyQ toxicity in cis can also do so in trans. Furthermore, the prion conformation of the yeast protein Rnq1 and the level of expression of a suite of other glutamine-rich proteins profoundly affect polyQ toxicity. They can convert polyQ expansion proteins from toxic to benign and vice versa. Our work presents a paradigm for how a complex, dynamic interplay between intramolecular features of polyQ proteins and intermolecular factors in the cellular environment might determine the unique pathobiologies of polyQ expansion proteins.huntingtin ͉ Huntington's disease ͉ protein misfolding ͉ yeast A variety of human diseases are characterized by the accumulation of aggregated, misfolded proteins (1, 2). In some cases, such as cystic fibrosis, disease is caused by the loss of a vital protein function due simply to this misfolding and aggregation. In other cases, however, protein misfolding creates novel, toxic properties. For example, in Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) disorders, misfolded proteins disrupt proper cellular function and cause cytotoxicity (3,4). In all of these diseases the amino acid sequences of the particular misfolded protein and their interactions determined by its specific environment must govern toxicity. Yet, despite intense research, we know little about these intramolecular and intermolecular factors. Without identifying and understanding these factors at a molecular level it will be difficult to devise the most effective therapeutic strategies to treat protein misfolding diseases.The polyQ diseases present a promising starting point to study this very general problem. In all of these diseases polyQ expansions constitute the molecular basis of disease (5). However, the disease pathologies (including the cell types most strongly affected) and the individual proteins that bear the disease-causing polyQ expansions are very distinct (5). Consequently, the amino acids flanking the polyQ region and the sets of cellular factors that specifically interact with each polyQ expansion protein are different. Combinations of these factors must account for their different pathobiologies. Recent studies have started to identify protein-protein and genetic interaction networks for polyQ proteins (6-8), but the current data do not yet ...

The authors define how small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial prion, and then they exploit this knowledge to identify the human amyloid depolymerase.