Mutation processes at the protein level: is Lamarck back?

The experimental evidence accumulated for the last half of the century clearly suggests that inherited variation is not restricted to the changes in genomic sequences. The prion model, originally based on unusual transmission of certain neurodegenerative diseases in mammals, provides a molecular mechanism for the template-like reproduction of alternative protein conformations. Recent data extend this model to protein-based genetic elements in yeast and other fungi. Reproduction and transmission of yeast protein-based genetic elements is controlled by the "prion replication" machinery of the cell, composed of the protein helpers responsible for the processes of assembly and disassembly of protein structures and multiprotein complexes. Among these, the stress-related chaperones of Hsp100 and Hsp70 groups play an important role. Alterations of levels or activity of these proteins result in "mutator" or "antimutator" affects in regard to protein-based genetic elements. "Protein mutagens" have also been identified that affect formation and/or propagation of the alternative protein conformations. Prion-forming abilities appear to be conserved in evolution, despite the divergence of the corresponding amino acid sequences. Moreover, a wide variety of proteins of different origins appear to possess the ability to form amyloid-like aggregates, that in certain conditions might potentially result in prion-like switches. This suggests a possible mechanism for the inheritance of acquired traits, postulated in the Lamarckian theory of evolution. The prion model also puts in doubt the notion that cloned animals are genetically identical to their genome donors, and suggests that genome sequence would not provide a complete information about the genetic makeup of an organism.     

Newly identified prions in budding yeast, and their possible functions

Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we summarize the characteristics of each prion element, and discuss their potential functional roles in yeast biology.     

Analysis of yeast prion aggregates with amyloid-staining compound in vivo

Yeast prions are protein-based genetic elements whose non-Mendelian patterns of inheritance are explained by their inheritance of altered conformations. Here we showed that aggregates made by overexpression of two different prion domains of Sup35 and Rnq1, were stained in yeast by thioflavin-S, an amyloid binding compound. These results suggested that yeast prion domains take the form of amyloid in vivo, and supported the idea that the self-propagating property of amyloids is responsible for the heritable traits of yeast prions.     

Destruction or potentiation of different prions catalyzed by similar Hsp104 remodeling activities

Yeast prions are protein-based genetic elements that self-perpetuate changes in protein conformation and function. A protein-remodeling factor, Hsp104, controls the inheritance of several yeast prions, including those formed by Sup35 and Ure2. Perplexingly, deletion of Hsp104 eliminates Sup35 and Ure2 prions, whereas overexpression of Hsp104 purges cells of Sup35 prions, but not Ure2 prions. Here, we used pure components to dissect how Hsp104 regulates prion formation, growth, and division. For both Sup35 and Ure2, Hsp104 catalyzes de novo prion nucleation from soluble, native protein. Using a distinct mechanism, Hsp104 fragments both prions to generate new prion assembly surfaces. For Sup35, the fragmentation endpoint is an ensemble of noninfectious, amyloid-like aggregates and soluble protein that cannot replicate conformation. In vivid distinction, the endpoint of Ure2 fragmentation is short prion fibers with enhanced infectivity and self-replicating ability. These advances explain the distinct effects of Hsp104 on the inheritance of the two prions.     

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 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.     

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.     

Evidence for a protein mutator in yeast: role of the Hsp70-related chaperone ssb in formation, stability, and toxicity of the [PSI] prion

Propagation of the yeast protein-based non-Mendelian element [PSI], a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70. While overproduction of Hsp104 protein cures cells of [PSI], overproduction of the Ssa1 protein of the Hsp70 family protects [PSI] from the curing effect of Hsp104. Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on [PSI] which are opposite those of Ssa. Ssb overproduction increases, while Ssb depletion decreases, [PSI] curing by the overproduced Hsp104. Both spontaneous [PSI] formation and [PSI] induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb. This is the first example when inactivation of an unrelated cellular protein facilitates prion formation. Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance. Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the [PSI]-curing chemical agent guanidine hydrochloride. Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates.     

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.     

The Genetic Control of the Formation and Propagation of the [PSI+] Prion of Yeast

It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI+] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learned much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI+] prion.     

The role of conformational flexibility in prion propagation and maintenance for Sup35p.

The [PSI(+)] factor of Saccharomyces cerevisiae is a protein-based genetic element (prion) comprised of a heritable altered conformation of the cytosolic translation termination factor Sup35p. In vitro, the prion-determining region (NM) of Sup35p undergoes conformational conversion from a highly flexible soluble state to structured amyloid fibers, with a rate that is greatly accelerated by preformed NM fiber nuclei. Nucleated conformational conversion is the molecular basis of the genetic inheritance of [PSI(+)] and provides a new model for studying amyloidogenesis. Here we investigate the importance of structure and structural flexibility in soluble NM. Elevated temperatures, chemical chaperones and certain mutations in NM increase or change its structural content and inhibit or enhance nucleated conformational conversion. We propose that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.     

Prion proteostasis

Infectious amyloid forms of the release factor, Sup35, comprise the yeast prion [PSI⁺]. This protein-based unit of inheritance is an evolutionary capacitor able to release cryptic genetic variation during environmental stress and generate potentially beneficial phenotypes. Genetic data have uncovered a sophisticated proteostasis network that tightly regulates [PSI⁺] formation, propagation and elimination. Central to this network, is the AAA+ ATPase and protein disaggregase, Hsp104. Shifting the balance of the cytosolic Hsp70:Hsp40 chaperone machinery and associated nucleotide exchange factors also influences the [PSI⁺] prion cycle. Yet, a precise understanding of how these systems co-operate to directly modulate the protein folding events required for sustainable Sup35 prionogenesis has remained elusive. Here, we spotlight recent advances that begin to clarify this issue. We suggest that the Hsp70:Hsp40 chaperone machinery functions collectively as a rheostat that adjusts Hsp104’s basic prion-remodeling activities.     

Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.     

Large-scale de novo prediction of physical protein-protein association

Information about the physical association of proteins is extensively used for studying cellular processes and disease mechanisms. However, complete experimental mapping of the human interactome will remain prohibitively difficult in the near future. Here we present a map of predicted human protein interactions that distinguishes functional association from physical binding. Our network classifies more than 5 million protein pairs predicting 94,009 new interactions with high confidence. We experimentally tested a subset of these predictions using yeast two-hybrid analysis and affinity purification followed by quantitative mass spectrometry. Thus we identified 462 new protein-protein interactions and confirmed the predictive power of the network. These independent experiments address potential issues of circular reasoning and are a distinctive feature of this work. Analysis of the physical interactome unravels subnetworks mediating between different functional and physical subunits of the cell. Finally, we demonstrate the utility of the network for the analysis of molecular mechanisms of complex diseases by applying it to genome-wide association studies of neurodegenerative diseases. This analysis provides new evidence implying TOMM40 as a factor involved in Alzheimer's disease. The network provides a high-quality resource for the analysis of genomic data sets and genetic association studies in particular. Our interactome is available via the hPRINT web server at: www.print-db.org.     

Prion Propagation: The Role of Protein Dynamics

The transfer of phenotypes from one individual to another is a fundamental aspect of biology. In addition to traditional nucleic acid-based genetic determinants, unique proteins known as prions can also act as elements of inheritance, infectivity, and disease. Nucleic acids and proteins encode genetic information in distinct ways, either in the sequence of bases in DNA or RNA or in the three dimensional structure of the polypeptide chain. Given these differences in the nature of the genetic repository, the mechanisms underlying the transmission of nucleic acid-based and protein-based phenotypes are necessarily distinct. While the appearance, persistence and transfer of nucleic acid determinants require the synthesis of new polymers, recent studies indicate that prions are propagated through dynamic transitions in the structure of existing protein.     

"Prion-proof" for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+

Prions are self-propagating, infectious protein conformations. The mammalian prion, PrP(Sc), responsible for neurodegenerative diseases like bovine spongiform encephalopathy (BSE; "mad cow" disease) and Creutzfeldt-Jakob's disease, appears to be a beta-sheet-rich amyloid conformation of PrP(c) that converts PrP(c) into PrP(Sc). However, an unequivocal demonstration of "protein-only" infection by PrP(Sc) is still lacking. So far, protein only infection has been proven for three prions, [PSI(+)], [URE3] and [Het-s], all of fungal origin. Considerable evidence supports the hypothesis that another protein, the yeast Rnq1p, can form a prion, [PIN(+)]. While Rnq1p does not lose any known function upon prionization, [PIN(+)] has interesting positive phenotypes: facilitating the appearance and destabilization of other prions as well as the aggregation of polyglutamine extensions of the Huntingtin protein. Here, we polymerize a Gln/Asn-rich recombinant fragment of Rnq1p into beta-sheet-rich amyloid-like aggregates. While the method used for [PSI(+)] and [URE3] infectivity assays did not yield protein-only infection for the Rnq1p aggregates, we did successfully obtain protein-only infection by modifying the protocol. This work proves that [PIN(+)] is a prion mediated by amyloid-like aggregates of Rnq1p, and supports the hypothesis that heterologous prions affect each other's appearance and propagation through interaction of their amyloid-like regions.     

Cloning strategy for producing brush-forming protein-based polymers

Brush-forming polymers are being used in a variety of applications, and by using recombinant DNA technology, there exists the potential to produce protein-based polymers that incorporate unique structures and functions in these brush layers. Despite this potential, production of protein-based brush-forming polymers is not routinely performed. For the design and production of new protein-based polymers with optimal brush-forming properties, it would be desirable to have a cloning strategy that allows an iterative approach wherein the protein based-polymer product can be produced and evaluated, and then if necessary, it can be sequentially modified in a controlled manner to obtain optimal surface density and brush extension. In this work, we report on the development of a cloning strategy intended for the production of protein-based brush-forming polymers. This strategy is based on the assembly of modules of DNA that encode for blocks of protein-based polymers into a commercially available expression vector; there is no need for custom-modified vectors and no need for intermediate cloning vectors. Additionally, because the design of new protein-based biopolymers can be an iterative process, our method enables sequential modification of a protein-based polymer product. With at least 21 bacterial expression vectors and 11 yeast expression vectors compatible with this strategy, there are a number of options available for production of protein-based polymers. It is our intent that this strategy will aid in advancing the production of protein-based brush-forming polymers.     

The [PSI+] prion of yeast: a problem of inheritance

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.     

Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.     

The Genetic Control of the Formation and Propagation of the [PSI+] Prion of Yeast

It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI⁺] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learnt much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this Chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI⁺] prion.Key Words: yeast, [PSI], prion, SUP35/eRF3, SUP45/eRF1, antisuppressor, [PSI⁺] maintenance genes, Hsp104, prion antagonists