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In JoVE (1)
Other Publications (10)
- Proceedings of the National Academy of Sciences of the United States of America
- Proceedings of the National Academy of Sciences of the United States of America
- Proceedings of the National Academy of Sciences of the United States of America
- Proceedings of the National Academy of Sciences of the United States of America
- Genes & Development
- Nature Chemical Biology
- Prion
- Methods (San Diego, Calif.)
- The Journal of Biological Chemistry
- Prion
Articles by Martin L. Duennwald in JoVE
JoVE General
Growth Assays to Assess Polyglutamine Toxicity in Yeast
Boston Biomedical Research Institute
This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.
Other articles by Martin L. Duennwald on PubMed
A Network of Protein Interactions Determines Polyglutamine Toxicity
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.
Flanking Sequences Profoundly Alter Polyglutamine Toxicity in Yeast
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 approximately 39 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.
Chaperone-dependent Amyloid Assembly Protects Cells from Prion Toxicity
Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] 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.
Direct and Selective Elimination of Specific Prions and Amyloids by 4,5-dianilinophthalimide and Analogs
Mechanisms to safely eliminate amyloids and preamyloid oligomers associated with many devastating diseases are urgently needed. Biophysical principles dictate that small molecules are unlikely to perturb large intermolecular protein-protein interfaces, let alone extraordinarily stable amyloid interfaces. Yet 4,5-dianilinophthalimide (DAPH-1) reverses Abeta42 amyloidogenesis and neurotoxicity, which is associated with Alzheimer's disease. Here, we show that DAPH-1 and select derivatives are ineffective against several amyloidogenic proteins, including tau, alpha-synuclein, Ure2, and PrP, but antagonize the yeast prion protein, Sup35, in vitro and in vivo. This allowed us to exploit several powerful new tools created for studying the conformational transitions of Sup35 and decipher the mechanisms by which DAPH-1 and related compounds antagonize the prion state. During fibrillization, inhibitory DAPHs alter the folding of Sup35's amyloidogenic core, preventing amyloidogenic oligomerization and specific recognition events that nucleate prion assembly. Select DAPHs also are capable of attacking preformed amyloids. They remodel Sup35 prion-specific intermolecular interfaces to create morphologically altered aggregates with diminished infectivity and self-templating activity. Our studies provide mechanistic insights and reinvigorate hopes for small-molecule therapies that specifically disrupt intermolecular amyloid contacts.
Impaired ERAD and ER Stress Are Early and Specific Events in Polyglutamine Toxicity
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.
A Synergistic Small-molecule Combination Directly Eradicates Diverse Prion Strain Structures
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 intramolecular 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.
Countering Amyloid Polymorphism and Drug Resistance with Minimal Drug Cocktails
Several fatal, progressive neurodegenerative diseases, including various prion and prion-like disorders, are connected with the misfolding of specific proteins. These proteins misfold into toxic oligomeric species and a spectrum of distinct self-templating amyloid structures, termed strains. Hence, small molecules that prevent or reverse these protein-misfolding events might have therapeutic utility. Yet it is unclear whether a single small molecule can antagonize the complete repertoire of misfolded forms encompassing diverse amyloid polymorphs and soluble oligomers. We have begun to investigate this issue using the yeast prion protein Sup35 as an experimental paradigm. We have discovered that a polyphenol, (-)epigallocatechin-3-gallate (EGCG), effectively inhibited the formation of infectious amyloid forms (prions) of Sup35 and even remodeled preassembled prions. Surprisingly, EGCG selectively modulated specific prion strains and even selected for EGCG-resistant prion strains with novel structural and biological characteristics. Thus, treatment with a single small molecule antagonist of amyloidogenesis can select for novel, drug-resistant amyloid polymorphs. Importantly, combining EGCG with another small molecule, 4,5-bis-(4-methoxyanilino)phthalimide, synergistically antagonized and remodeled a wide array of Sup35 prion strains without producing any drug-resistant prions. We suggest that minimal drug cocktails, small collections of drugs that collectively antagonize all amyloid polymorphs, should be identified to besiege various neurodegenerative disorders.
Monitoring Polyglutamine Toxicity in Yeast
Experiments in yeast have significantly contributed to our understanding of general aspects of biochemistry, genetics, and cell biology. Yeast models have also delivered deep insights in to the molecular mechanism underpinning human diseases, including neurodegenerative diseases. Many neurodegenerative diseases are associated with the conversion of a protein from a normal and benign conformation into a disease-associated and toxic conformation - a process called protein misfolding. The misfolding of proteins with abnormally expanded polyglutamine (polyQ) regions causes several neurodegenerative diseases, such as Huntington's disease and the Spinocerebellar Ataxias. Yeast cells expressing polyQ expansion proteins recapitulate polyQ length-dependent aggregation and toxicity, which are hallmarks of all polyQ-expansion diseases. The identification of modifiers of polyQ toxicity in yeast revealed molecular mechanisms and cellular pathways that contribute to polyQ toxicity. Notably, several of these findings in yeast were reproduced in other model organisms and in human patients, indicating the validity of the yeast polyQ model. Here, we describe different expression systems for polyQ-expansion proteins in yeast and we outline experimental protocols to reliably and quantitatively monitor polyQ toxicity in yeast.
Ordered Assembly of Heat Shock Proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on Expanded Polyglutamine Fragments Revealed by Chemical Probes
In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.
Polyglutamine Misfolding in Yeast: Toxic and Protective Aggregation
Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.
