For several decades scientists have speculated that the key to understanding age-related neurodegenerative disorders may be found in the unusual biology of the prion diseases. Recently, owing largely to the advent of new disease models, this hypothesis has gained experimental momentum. In a remarkable variety of diseases, specific proteins have been found to misfold and aggregate into seeds that structurally corrupt like proteins, causing them to aggregate and form pathogenic assemblies ranging from small oligomers to large masses of amyloid. Proteinaceous seeds can therefore serve as self-propagating agents for the instigation and progression of disease. Alzheimer's disease and other cerebral proteopathies seem to arise from the de novo misfolding and sustained corruption of endogenous proteins, whereas prion diseases can also be infectious in origin. However, the outcome in all cases is the functional compromise of the nervous system, because the aggregated proteins gain a toxic function and/or lose their normal function. As a unifying pathogenic principle, the prion paradigm suggests broadly relevant therapeutic directions for a large class of currently intractable diseases.

Self-propagation of pathogenic protein aggregates in neurodegenerative diseases

https://www.med.upenn.edu/shorterlab/Papers/Member%20Papers/BrainRes-2012-King.pdf

The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease

INTRODUCTION The formation of dynamic, membraneless compartments using intracellular phase transitions such as phase separation and gelation provides an efficient way for cells to respond to environmental changes. Recent work has identified a special class of intrinsically disordered domains enriched for polar amino acids such as glycine, glutamine, serine, or tyrosine as potential drivers of phase separation in cells. However, more traditional work has highlighted the ability of these domains to drive the formation of fibrillar aggregates. Such domains are also known as prion domains. They have first been identified in budding yeast proteins that form amyloid-like aggregates. Because these aggregates are heritable and change the activity of the prion-domain–containing protein, they are thought to be a common mechanism for phenotypic inheritance in fungi and other organisms. However, the aggregation of prion domains has also been associated with neurodegenerative diseases in mammals. Therefore, the relationship between the role of these domains as drivers of phase separation and their ability to form prion-like aggregates is unknown. RATIONALE The budding yeast translation termination factor Sup35 is an archetypal prion-domain–containing protein. Sup35 forms irreversible heritable aggregates, and these aggregates have been proposed to be either a disease or an adaptation that generates heritable phenotypic variation in populations of budding yeast. Despite having been described almost 25 years ago, the physiological functions of the Sup35 prion domain and other prion-like domains remain unclear. Uncovering these functions is a prerequisite for understanding the evolutionary pressures shaping prion-like sequences and how their physiological and pathological transitions affect cellular fitness. RESULTS Here, we show that the prion domain of Sup35 drives the reversible phase separation of the translation termination factor into biomolecular condensates. These condensates are distinct and different from fibrillar amyloid-like prion particles. Combining genetic analysis in cells with in vitro reconstitution protein biochemistry and quantitative biophysical methods, we demonstrate that Sup35 condensates form by pH-induced liquid-like phase separation as a response to sudden stress. The condensates are liquid-like initially but subsequently solidify to form protective protein gels. Cryo–electron tomography demonstrates that these gel-like condensates consist of cross-linked Sup35 molecules forming a porous meshwork. A cluster of negatively charged amino acids functions as a pH sensor and regulates condensate formation. The ability to form biomolecular condensates is shared among distantly related budding yeast and fission yeast. This suggests that condensate formation is a conserved and ancestral function of the prion domain of Sup35. In agreement with an important physiological function of the prion domain, the catalytic guanosine triphosphatase (GTPase) domain of the translation termination factor Sup35 readily forms irreversible aggregates in the absence of the prion domain. Consequently, cells lacking the prion domain exhibit impaired translational activity and a growth defect when recovering from stress. These data demonstrate that the prion domain rescues the essential GTPase domain of Sup35 from irreversible aggregation, thus ensuring that the translation termination factor remains functional during harsh environmental conditions. CONCLUSION The prion domain of Sup35 is a highly regulated molecular device that has the ability to sense and respond to physiochemical changes within cells. The N-terminal prion domain provides the interactions that drive liquid phase separation. Phase separation is regulated by the adjacent stress sensor. The synergy of these two modules enables the essential translation termination factor to rapidly form protective condensates during stress. This suggests that prion domains are protein-specific stress sensors and modifiers of protein phase transitions that allow cells to respond to specific environmental conditions.

Phase separation of a yeast prion protein promotes cellular fitness

Amyloid-like Self-Assembly of a Cellular Compartment

The prion paradigm has emerged as a unifying molecular principle for the pathogenesis of many age-related neurodegenerative diseases. This paradigm holds that a fundamental cause of specific disorders is the misfolding and seeded aggregation of certain proteins. The concept arose from the discovery that devastating brain diseases called spongiform encephalopathies are transmissible to new hosts by agents consisting solely of a misfolded protein, now known as the prion protein. Accordingly, "prion" was defined as a "proteinaceous infectious particle." As the concept has expanded to include other diseases, many of which are not infectious by any conventional definition, the designation of prions as infectious agents has become problematic. We propose to define prions as "proteinaceous nucleating particles" to highlight the molecular action of the agents, lessen unwarranted apprehension about the transmissibility of noninfectious proteopathies, and promote the wider acceptance of this revolutionary paradigm by the biomedical community.

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Neurodegenerative Diseases: Expanding the Prion Concept

A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.

Yeast Prions: Structure, Biology, and Prion-Handling Systems

The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-(13)C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼05-nm distance between labels diagnostic of the in-register parallel amyloid architecture The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions

https://www.pnas.org/content/pnas/111/43/E4615.full.pdf

Locating folds of the in-register parallel β-sheet of the Sup35p prion domain infectious amyloid

The prions (infectious proteins) of Saccharomyces cerevisiae are proteins acting as genes, by templating their conformation from one molecule to another in analogy to DNA templating its sequence. Most yeast prions are amyloid forms of normally soluble proteins, and a single protein sequence can have any of several self-propagating forms (called prion strains or variants), analogous to the different possible alleles of a DNA gene. A central issue in prion biology is the structural basis of this conformational templating process. The in-register parallel β sheet structure found for several infectious yeast prion amyloids naturally suggests an explanation for this conformational templating. While most prions are plainly diseases, the [Het-s] prion of Podospora anserina may be a functional amyloid, with important structural implications. Yeast prions are important models for human amyloid diseases in general, particularly because new evidence is showing infectious aspects of several human amyloidoses not prev...

/pdf/amyloids-and-yeast-prion-biology-2cq1n6one8.pdf

Amyloids and Yeast Prion Biology

The yeast prions [URE3] and [PSI] are not found in wild strains, suggesting they are not an advantage. Prion-forming ability is not conserved, even within Saccharomyces, suggesting it is a disease. Prion domains have non-prion functions, explaining some conservation of sequence. However, in spite of the sequence being constrained in evolution by these non-prion functions, the prion domains vary more rapidly than the remainder of the molecule, and these changes produce a transmission barrier, suggesting that these changes were selected to block prion infection. Yeast prions [PSI] and [URE3] induce a cellular stress response (Hsp104 and Hsp70 induction), suggesting the cells are not happy about being infected. Recently, we showed that the array of [PSI] and [URE3] prions includes a majority of lethal or very toxic variants, a result not expected if either prion were an adaptive cellular response to stress.

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases.

Overproduction or deficiency of many chaperones and other cellular components cure the yeast prions [PSI+] (formed by Sup35p) or [URE3] (based on Ure2p). However, at normal expression levels, Btn2p and Cur1p eliminate most newly arising [URE3] variants but do not cure [PSI+], even after overexpression. Deficiency or overproduction of Hsp104 cures the [PSI+] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [PSI+], but cannot cure it by overproduction, thus separating filament cleavage from curing activities. Here we show that most [PSI+] variants arising spontaneously in an hsp104(T160M) strain are cured by restoration of just normal levels of the WT Hsp104. Both strong and weak [PSI+] variants are among those cured by this process. This normal-level Hsp104 curing is promoted by Sti1p, Hsp90, and Sis1p, proteins previously implicated in the Hsp104 overproduction curing of [PSI+]. The [PSI+] prion arises in hsp104(T160M) cells at more than 10-fold the frequency in WT cells. The curing activity of Hsp104 thus constitutes an antiprion system, culling many variants of the [PSI+] prion at normal Hsp104 levels.

https://www.pnas.org/content/pnas/114/21/E4193.full.pdf

Anton Gorkovskiy

Papers

Yeast Prions: Structure, Biology, and Prion-Handling Systems

Locating folds of the in-register parallel β-sheet of the Sup35p prion domain infectious amyloid

Amyloids and Yeast Prion Biology

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases.

Hsp104 disaggregase at normal levels cures many [PSI+] prion variants in a process promoted by Sti1p, Hsp90, and Sis1p.