Kevin C. Stein

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

BackgroundMutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.ResultsWe quantified significant variation among C. briggsae isolates for all phenotypes measured, only some of which was statistically associated with isolate-specific ND5 deletion frequency. We found that fecundity-related traits and pharyngeal pumping rate were strongly inversely related to ND5 deletion level and that C. briggsae isolates with high ND5 deletion levels experienced a tradeoff between early fecundity and lifespan. Conversely, oxidative stress resistance was only weakly associated with ND5 deletion level while ATP content was unrelated to deletion level. Finally, mean levels of reactive oxygen species measured in vivo showed a significant non-linear relationship with ND5 deletion level, a pattern that may be driven by among-isolate variation in antioxidant or other compensatory mechanisms.ConclusionsOur findings suggest that the ND5 deletion may adversely affect fitness and mitochondrial functioning while promoting aging in natural populations, and help to further establish this species as a useful model for explicit tests of hypotheses in aging biology and mitochondrial genetics.

Selective sweeps and parallel mutation in the adaptive recovery from deleterious mutation in Caenorhabditis elegans.

Deleterious mutation poses a serious threat to human health and the persistence of small populations. Although adaptive recovery from deleterious mutation has been well-characterized in prokaryotes, the evolutionary mechanisms by which multicellular eukaryotes recover from deleterious mutation remain unknown. We applied high-throughput DNA sequencing to characterize genomic divergence patterns associated with the adaptive recovery from deleterious mutation using a Caenorhabditis elegans recovery-line system. The C. elegans recovery lines were initiated from a low-fitness mutation-accumulation (MA) line progenitor and allowed to independently evolve in large populations (N ∼ 1000) for 60 generations. All lines rapidly regained levels of fitness similar to the wild-type (N2) MA line progenitor. Although there was a near-zero probability of a single mutation fixing due to genetic drift during the recovery experiment, we observed 28 fixed mutations. Cross-generational analysis showed that all mutations went from undetectable population-level frequencies to a fixed state in 10-20 generations. Many recovery-line mutations fixed at identical timepoints, suggesting that the mutations, if not beneficial, hitchhiked to fixation during selective sweep events observed in the recovery lines. No MA line mutation reversions were detected. Parallel mutation fixation was observed for two sites in two independent recovery lines. Analysis using a C. elegans interactome map revealed many predicted interactions between genes with recovery line-specific mutations and genes with previously accumulated MA line mutations. Our study suggests that recovery-line mutations identified in both coding and noncoding genomic regions might have beneficial effects associated with compensatory epistatic interactions.

The [RNQ+] prion: A model of both functional and pathological amyloid

The formation of fibrillar amyloid is most often associated with protein conformational disorders such as prion diseases, Alzheimer disease and Huntington disease. Interestingly, however, an increasing number of studies suggest that amyloid structures can sometimes play a functional role in normal biology. Several proteins form self-propagating amyloids called prions in the budding yeast Saccharomyces cerevisiae. These unique elements operate by creating a reversible, epigenetic change in phenotype. While the function of the non-prion conformation of the Rnq1 protein is unclear, the prion form, [RNQ+], acts to facilitate the de novo formation of other prions to influence cellular phenotypes. The [RNQ+] prion itself does not adversely affect the growth of yeast, but the overexpression of Rnq1p can form toxic aggregated structures that are not necessarily prions. The [RNQ+] prion is also involved in dictating the aggregation and toxicity of polyglutamine proteins ectopically expressed in yeast. Thus, the [RNQ+] prion provides a tractable model that has the potential to reveal significant insight into the factors that dictate how amyloid structures are initiated and propagated in both physiological and pathological contexts.

Prion strains and amyloid polymorphism influence phenotypic variation.

The deposition of protein aggregates is a unifying feature of a large class of diseases known as protein conformational disorders, which includes Alzheimer disease and prion diseases. One of the most fascinating and puzzling aspects of such diseases is the phenomenon of amyloid polymorphism, whereby a single disease-associated protein forms different types of ordered aggregate structures. This is best exemplified in prion diseases, in which these different structures, called prion strains, are responsible for much of the variation in pathology and disease transmission. Here, we review the current knowledge of prion strains and amyloid polymorphism, highlighting how diversity in amyloid structure relates to phenotypic differences.

Extensive diversity of prion strains is defined by differential chaperone interactions and distinct amyloidogenic regions.

Amyloidogenic proteins associated with a variety of unrelated diseases are typically capable of forming several distinct self-templating conformers. In prion diseases, these different structures, called prion strains (or variants), confer dramatic variation in disease pathology and transmission. Aggregate stability has been found to be a key determinant of the diverse pathological consequences of different prion strains. Yet, it remains largely unclear what other factors might account for the widespread phenotypic variation seen with aggregation-prone proteins. Here, we examined a set of yeast prion variants of the [RNQ+] prion that differ in their ability to induce the formation of another yeast prion called [PSI+]. Remarkably, we found that the [RNQ+] variants require different, non-contiguous regions of the Rnq1 protein for both prion propagation and [PSI+] induction. This included regions outside of the canonical prion-forming domain of Rnq1. Remarkably, such differences did not result in variation in aggregate stability. Our analysis also revealed a striking difference in the ability of these [RNQ+] variants to interact with the chaperone Sis1. Thus, our work shows that the differential influence of various amyloidogenic regions and interactions with host cofactors are critical determinants of the phenotypic consequences of distinct aggregate structures. This helps reveal the complex interdependent factors that influence how a particular amyloid structure may dictate disease pathology and progression.

Spontaneous Variants of the (RNQ+) Prion in Yeast Demonstrate the Extensive Conformational Diversity Possible with Prion Proteins

Prion strains (or variants) are structurally distinct amyloid conformations arising from a single polypeptide sequence. The existence of prion strains has been well documented in mammalian prion diseases. In many cases, prion strains manifest as variation in disease progression and pathology, and in some cases, these prion strains also show distinct biochemical properties. Yet, the underlying basis of prion propagation and the extent of conformational possibilities available to amyloidogenic proteins remain largely undefined. Prion proteins in yeast that are also capable of maintaining multiple self-propagating structures have provided much insight into prion biology. Here, we explore the vast structural diversity of the yeast prion [RNQ+] in Saccharomyces cerevisiae. We screened for the formation of [RNQ+] in vivo, allowing us to calculate the rate of spontaneous formation as ~2.96x10-6, and successfully isolate several different [RNQ+] variants. Through a comprehensive set of biochemical and biological analyses, we show that these prion variants are indeed novel. No individual property or set of properties, including aggregate stability and size, was sufficient to explain the physical basis and range of prion variants and their resulting cellular phenotypes. Furthermore, all of the [RNQ+] variants that we isolated were able to facilitate the de novo formation of the yeast prion [PSI+], an epigenetic determinant of translation termination. This supports the hypothesis that [RNQ+] acts as a functional amyloid in regulating the formation of [PSI+] to produce phenotypic diversity within a yeast population and promote adaptation. Collectively, this work shows the broad spectrum of available amyloid conformations, and thereby expands the foundation for studying the complex factors that interact to regulate the propagation of distinct aggregate structures.

Myopathy-causing Mutations in an HSP40 Chaperone Disrupt Processing of Specific Client Conformers

Background: Mutations in the G/F domain of the human HSP40 chaperone DNAJB6 cause a protein aggregate myopathy. Results: Homologous mutations in the yeast HSP40 Sis1 alter prion propagation in a prion strain-dependent manner. Conclusion: G/F domain mutations affect chaperone-mediated processing of particular client conformers. Significance: Impaired processing of certain substrate conformations may be one mechanism that contributes to chaperonopathy pathogenesis. The molecular chaperone network protects against the toxic misfolding and aggregation of proteins. Disruption of this network leads to a variety of protein conformational disorders. One such example recently discovered is limb-girdle muscular dystrophy type 1D (LGMD1D), which is caused by mutation of the HSP40 chaperone DNAJB6. All LGMD1D-associated mutations localize to the conserved G/F domain of DNAJB6, but the function of this domain is largely unknown. Here, we exploit the yeast HSP40 Sis1, which has known aggregation-prone client proteins, to gain insight into the role of the G/F domain and its significance in LGMD1D pathogenesis. Strikingly, we demonstrate that LGMD1D mutations in a Sis1-DNAJB6 chimera differentially impair the processing of specific conformers of two yeast prions, [RNQ+] and [PSI+]. Importantly, these differences do not simply correlate to the sensitivity of these prion strains to changes in chaperone levels. Additionally, we analyzed the effect of LGMD1D-associated DNAJB6 mutations on TDP-43, a protein known to form inclusions in LGMD1D. We show that the DNAJB6 G/F domain mutants disrupt the processing of nuclear TDP-43 stress granules in mammalian cells. These data suggest that the G/F domain mediates chaperone-substrate interactions in a manner that extends beyond recognition of a particular client and to a subset of client conformers. We propose that such selective chaperone disruption may lead to the accumulation of toxic aggregate conformers and result in the development of LGMD1D and perhaps other protein conformational disorders.

Structural variants of yeast prions show conformer‐specific requirements for chaperone activity

Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1 and its human orthologue Hdj1 had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone‐client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation.

Regulation of the Hsp104 Middle Domain Activity Is Critical for Yeast Prion Propagation

Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.

Heterologous prion-forming proteins interact to cross-seed aggregation in Saccharomyces cerevisiae

The early stages of protein misfolding remain incompletely understood, as most mammalian proteinopathies are only detected after irreversible protein aggregates have formed. Cross-seeding, where one aggregated protein templates the misfolding of a heterologous protein, is one mechanism proposed to stimulate protein aggregation and facilitate disease pathogenesis. Here, we demonstrate the existence of cross-seeding as a crucial step in the formation of the yeast prion [PSI+], formed by the translation termination factor Sup35. We provide evidence for the genetic and physical interaction of the prion protein Rnq1 with Sup35 as a predominant mechanism leading to self-propagating Sup35 aggregation. We identify interacting sites within Rnq1 and Sup35 and determine the effects of breaking and restoring a crucial interaction. Altogether, our results demonstrate that single-residue disruption can drastically reduce the effects of cross-seeding, a finding that has important implications for human protein misfolding disorders.