Tricia R. Serio

The prion hypothesis: from biological anomaly to basic regulatory mechanism

Prions are unusual proteinaceous infectious agents that are typically associated with a class of fatal degenerative diseases of the mammalian brain. However, the discovery of fungal prions, which are not associated with disease, suggests that we must now consider the effect of these factors on basic cellular physiology in a different light. Fungal prions are epigenetic determinants that can alter a range of cellular processes, including metabolism and gene expression pathways, and these changes can lead to a range of prion-associated phenotypes. The mechanistic similarities between prion propagation in mammals and fungi suggest that prions are not a biological anomaly but instead could be a newly appreciated and perhaps ubiquitous regulatory mechanism.

Hsp104-Dependent Remodeling of Prion Complexes Mediates Protein-Only Inheritance

Inheritance of phenotypic traits depends on two key events: replication of the determinant of that trait and partitioning of these copies between mother and daughter cells. Although these processes are well understood for nucleic acid–based genes, the mechanisms by which protein-only or prion-based genetic elements direct phenotypic inheritance are poorly understood. Here, we report a process crucial for inheritance of the Saccharomyces cerevisiae prion [PSI+], a self-replicating conformer of the Sup35 protein. By tightly controlling expression of a Sup35-GFP fusion, we directly observe remodeling of existing Sup35[PSI+] complexes in vivo. This dynamic change in Sup35[PSI+] is lost when the molecular chaperone Hsp104, a factor essential for propagation of all yeast prions, is functionally impaired. The loss of Sup35[PSI+] remodeling by Hsp104 decreases the mobility of these complexes in the cytosol, creates a segregation bias that limits their transmission to daughter cells, and consequently diminishes the efficiency of conversion of newly made Sup35 to the prion form. Our observations resolve several seemingly conflicting reports on the mechanism of Hsp104 action and point to a single Hsp104-dependent event in prion propagation.

Prion protein remodelling confers an immediate phenotypic switch

In a variety of systems, proteins have been linked to processes historically limited to nucleic acids, such as infectivity and inheritance. These atypical proteins, termed prions, lack sequence homology but are collectively defined by their capacity to adopt multiple physical and therefore functional states in vivo. Newly synthesized prion protein generally adopts the form already present in the cell, and this in vivo folding bias directs the near faithful transmission of the corresponding phenotypic state. Switches between the prion and non-prion phenotypes can occur in vivo; however, the fate of existing protein during these transitions and its effects on the emergence of new traits remain major unanswered questions. Here, we determine the changes in protein-state that induce phenotypic switching for the yeast prion Sup35/[PSI+]. We show that the prion form does not need to be specified by an alternate misfolding pathway initiated during Sup35 synthesis but instead can be accessed by mature protein. This remodelling of protein from one stable form to another is accompanied by the loss of Sup35 activity, evoking a rapid change in cellular phenotype within a single cell cycle.

Protein-only inheritance in yeast: something to get [PSI+]-ched about

Recent work suggests that two unrelated phenotypes, [PSI+] and [URE3], in the yeast Saccharomyces cerevisiae are transmitted by non-covalent changes in the physical states of their protein determinants, Sup35p and Ure2p, rather than by changes in the genes that encode these proteins. The mechanism by which alternative protein states are self-propagating is the key to understanding how proteins function as elements of epigenetic inheritance. Here, we focus on recent molecular-genetic analysis of the inheritance of the [PSI+] factor of S. cerevisiae. Insights into this process might be extendable to a group of mammalian diseases (the amyloidoses), which are also believed to be a manifestation of self-perpetuating changes in protein conformation.

A size threshold limits prion transmission and establishes phenotypic diversity

Processing Prion Phenotype How misfolding of a prion protein translates into transmissible changes in cellular physiology is unclear. Derdowski et al. (p. 680) integrated a computational model of prion aggregate dynamics with an empirical analysis of the physical and functional dynamics of prion protein in yeast cells. Remarkably, they found that prion phenotypes resulted from fluctuations in the accumulation of aggregates and suggested that it is the process rather than the product of protein misfolding that is crucial in establishing the severity or stability of the resulting phenotype. Yeast prion conformations specify phenotypes by affecting the size distribution of aggregates. According to the prion hypothesis, atypical phenotypes arise when a prion protein adopts an alternative conformation and persist when that form assembles into self-replicating aggregates. Amyloid formation in vitro provides a model for this protein-misfolding pathway, but the mechanism by which this process interacts with the cellular environment to produce transmissible phenotypes is poorly understood. Using the yeast prion Sup35/[PSI+], we found that protein conformation determined the size distribution of aggregates through its interactions with a molecular chaperone. Shifts in this range created variations in aggregate abundance among cells because of a size threshold for transmission, and this heterogeneity, along with aggregate growth and fragmentation, induced age-dependent fluctuations in phenotype. Thus, prion conformations may specify phenotypes as population averages in a dynamic system.

Yeast prion [psi +] and its determinant, Sup35p.

Publisher Summary [ PSI + ] and [ URE3 ] are two non-Mendelian genetic elements of the yeast Saccharomyces cerevisiae that appear to be inherited through an unusual mechanism—the continued propagation of an alternate protein conformation. The protein determinants of these elements, Sup35p for [ PSI + ] and Ure2p for [ URE3 ], have the unique ability to exist in at least two different, stable conformations in vivo . Although the spontaneous generation of one conformer is rare, this alternate form, once acquired, becomes predominant, influencing the other conformer to change states. This self-perpetuation of protein conformation is the key to the non-Mendelian inheritance of both [ PSI + ] and [ URE3 ]. In addition, the [ Het-S ] phenotype of Podospora anserina, another fungus, may be inherited by a similar mechanism. This chapter focuses on both in vivo and in vitro methods used to analyze [ PSI + ], the most extensively studied member of this group. The study of amyloidogenic proteins is complex both in vivo and in vitro. Each assay presented in this chapter provides unique information about the physical state of Sup35p, but these techniques also have inherent pitfalls.

Loss of amino-terminal acetylation suppresses a prion phenotype by modulating global protein folding.

N-terminal acetylation is among the most ubiquitous of protein modifications in eukaryotes. While loss of N-terminal acetylation is associated with many abnormalities, the molecular basis of these effects is known for only a few cases, where acetylation of single factors has been linked to binding avidity or metabolic stability. In contrast, the impact of N-terminal acetylation for the majority of the proteome, and its combinatorial contributions to phenotypes, are unknown. Here, by studying the yeast prion [PSI+], an amyloid of the Sup35 protein, we show that loss of N-terminal acetylation promotes general protein misfolding, a redeployment of chaperones to these substrates, and a corresponding stress response. These proteostasis changes, combined with the decreased stability of unacetylated Sup35 amyloid, reduce the size of prion aggregates and reverse their phenotypic consequences. Thus, loss of N-terminal acetylation, and its previously unanticipated role in protein biogenesis, globally resculpts the proteome to create a unique phenotype.

Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation

Protein misfolding underlies many neurodegenerative diseases, including the transmissible spongiform encephalopathies (prion diseases). Although cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, coexpression of dominant-negative prion mutants can overcome aggregate accumulation and disease progression through currently unknown pathways. Here we determine the mechanisms by which two mutants of the Saccharomyces cerevisiae Sup35 protein cure the [PSI+] prion. We show that both mutants incorporate into wild-type aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. Whereas wild-type aggregates are recalcitrant to cellular intervention, mixed aggregates are disassembled by the molecular chaperone Hsp104. Thus, rather than simply blocking misfolding, dominant-negative prion mutants target multiple events in aggregate biogenesis to enhance their susceptibility to endogenous quality-control pathways.

Prion dynamics and the quest for the genetic determinant in protein-only inheritance

According to the prion hypothesis, proteins may act in atypical roles as genetic elements of infectivity and inheritance by undergoing self-replicating changes in physical state. While the preponderance of evidence strongly supports this concept particularly in fungi, the detailed mechanisms by which distinct protein forms specify unique phenotypes are emerging concepts. A particularly active area of investigation is the molecular nature of the heritable species, which has been probed through genetic, biochemical, and cell biological experimentation as well as by mathematical modeling. Here, we suggest that these studies are converging to implicate small aggregates composed of prion-state conformers as the transmissible genetic determinants of protein-based phenotypes.

Amyloid-associated activity contributes to the severity and toxicity of a prion phenotype

The self-assembly of alternative conformations of normal proteins into amyloid aggregates has been implicated in both the acquisition of new functions and in the appearance and progression of disease. However, while these amyloidogenic pathways are linked to the emergence of new phenotypes, numerous studies have uncoupled the accumulation of aggregates from their biological consequences, revealing currently underappreciated complexity in the determination of these traits. Here, to explore the molecular basis of protein-only phenotypes, we focused on the S. cerevisiae Sup35/[PSI+] prion, which confers a translation termination defect and expression level-dependent toxicity in its amyloid form. Our studies reveal that aggregated Sup35 retains its normal function as a translation release factor. However, fluctuations in the composition and size of these complexes specifically alter the level of this aggregate-associated activity and thereby the severity and toxicity of the amyloid state. Thus, amyloid heterogeneity is a crucial contributor to protein-only phenotypes.