Randal Halfmann

A Systematic Survey Identifies Prions and Illuminates Sequence Features of Prionogenic Proteins

Prions are proteins that convert between structurally and functionally distinct states, one or more of which is transmissible. In yeast, this ability allows them to act as non-Mendelian elements of phenotypic inheritance. To further our understanding of prion biology, we conducted a bioinformatic proteome-wide survey for prionogenic proteins in S. cerevisiae, followed by experimental investigations of 100 prion candidates. We found an unexpected amino acid bias in aggregation-prone candidates and discovered that 19 of these could also form prions. At least one of these prion proteins, Mot3, produces a bona fide prion in its natural context that increases population-level phenotypic heterogeneity. The self-perpetuating states of these proteins present a vast source of heritable phenotypic variation that increases the adaptability of yeast populations to diverse environments.

Prions are a common mechanism for phenotypic inheritance in wild yeasts

The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare ‘diseases’ of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI+] or [MOT3+], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities.

Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation.

Pathogens and cellular danger signals activate sensors such as RIG-I and NLRP3 to produce robust immune and inflammatory responses through respective adaptor proteins MAVS and ASC, which harbor essential N-terminal CARD and PYRIN domains, respectively. Here, we show that CARD and PYRIN function as bona fide prions in yeast and that their prion forms are inducible by their respective upstream activators. Likewise, a yeast prion domain can functionally replace CARD and PYRIN in mammalian cell signaling. Mutations in MAVS and ASC that disrupt their prion activities in yeast also abrogate their ability to signal in mammalian cells. Furthermore, fibers of recombinant PYRIN can convert ASC into functional polymers capable of activating caspase-1. Remarkably, a conserved fungal NOD-like receptor and prion pair can functionally reconstitute signaling of NLRP3 and ASC PYRINs in mammalian cells. These results indicate that prion-like polymerization is a conserved signal transduction mechanism in innate immunity and inflammation.

Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits

Prions are an unusual form of epigenetics: Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid–associated changes. With intimate ties to protein homeostasis and a remarkable sensitivity to stress, prions are a robust mechanism that links environmental extremes with the acquisition and inheritance of new traits.

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.

Heritable Remodeling of Yeast Multicellularity by an Environmentally Responsive Prion

Prion proteins undergo self-sustaining conformational conversions that heritably alter their activities. Many of these proteins operate at pivotal positions in determining how genotype is translated into phenotype. But the breadth of prion influences on biology and their evolutionary significance are just beginning to be explored. We report that a prion formed by the Mot3 transcription factor, [MOT3(+)], governs the acquisition of facultative multicellularity in the budding yeast Saccharomyces cerevisiae. The traits governed by [MOT3(+)] involved both gains and losses of Mot3 regulatory activity. [MOT3(+)]-dependent expression of FLO11, a major determinant of cell-cell adhesion, produced diverse lineage-specific multicellular phenotypes in response to nutrient deprivation. The prions themselves were induced by ethanol and eliminated by hypoxia-conditions that occur sequentially in the natural respiro-fermentative cycles of yeast populations. These data demonstrate that prions can act as environmentally responsive molecular determinants of multicellularity and contribute to the natural morphological diversity of budding yeast.

Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms. Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity. However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE). This technique is finding widespread use for the detection and characterization of amyloid conformational variants. Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins. The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated. Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

Small-molecule activation of the TRAIL receptor DR5 in human cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates apoptosis through the death receptors DR4 and DR5. Because of its superior safety profile and high tumor specificity compared to other TNF family members, recombinant soluble TRAIL and agonistic antibodies against its receptors are actively being developed for clinical cancer therapy. Here, we describe the identification and characterization of the small molecules that directly target DR5 to initiate apoptosis in human cancer cells. The activity was initially discovered through a high-throughput chemical screen for compounds that promote cell death in synergy with a small-molecule mimetic of Smac, the antagonist for inhibitor of apoptosis protein. Structure-activity relationship studies yielded a more potent analog called bioymifi, which can act as a single agent to induce DR5 clustering and aggregation, leading to apoptosis. Thus, this study identified potential lead compounds for the development of small-molecule TRAIL mimics targeting DR5 for cancer therapy.

Prion formation by a yeast GLFG nucleoporin.

The self-assembly of proteins into higher order structures is both central to normal biology and a dominant force in disease. Certain glutamine/asparagine (Q/N)-rich proteins in the budding yeast Saccharomyces cerevisiae assemble into self-replicating amyloid-like protein polymers, or prions, that act as genetic elements in an entirely protein-based system of inheritance. The nuclear pore complex (NPC) contains multiple Q/N-rich proteins whose self-assembly has also been proposed to underlie structural and functional properties of the NPC. Here we show that an essential sequence feature of these proteins—repeating GLFG motifs—strongly promotes their self-assembly into amyloids with characteristics of prions. Furthermore, we demonstrate that Nup100 can form bona fide prions, thus establishing a previously undiscovered ability of yeast GLFG nucleoporins to adopt this conformational state in vivo.

Biochemical, Cell Biological, and Genetic Assays to Analyze Amyloid and Prion Aggregation in Yeast

Protein aggregates are associated with a variety of debilitating human diseases, but they can have functional roles as well. Both pathological and nonpathological protein aggregates display tremendous diversity, with substantial differences in aggregate size, morphology, and structure. Among the different aggregation types, amyloids are particularly remarkable, because of their high degree of order and their ability to form self-perpetuating conformational states. Amyloids form the structural basis for a group of proteins called prions, which have the ability to generate new phenotypes by a simple switch in protein conformation that does not involve changes in the sequence of the DNA. Although protein aggregates are notoriously difficult to study, recent technological developments and, in particular, the use of yeast prions as model systems, have been very instrumental in understanding fundamental aspects of aggregation. Here, we provide a range of biochemical, cell biological and yeast genetic methods that are currently used in our laboratory to study protein aggregation and the formation of amyloids and prions.