Eric D. Ross

Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Intrinsic protein disorder, amino acid composition, and histone terminal domains

Core and linker histones are the most abundant protein components of chromatin. Even though they lack intrinsic structure, the N-terminal “tail” domains (NTDs) of the core histones and the C-terminal tail domain (CTD) of linker histones bind to many different macromolecular partners while functioning in chromatin. Here we discuss the underlying physicochemical basis for how the histone terminal domains can be disordered and yet specifically recognize and interact with different macromolecules. The relationship between intrinsic disorder and amino acid composition is emphasized. We also discuss the potential structural consequences of acetylation and methylation of lysine residues embedded in intrinsically disordered histone tail domains.

Scrambled Prion Domains Form Prions and Amyloid

ABSTRACT The [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.

Prion domains: sequences, structures and interactions

Mammalian and most fungal infectious proteins (also known as prions) are self-propagating amyloid, a filamentous β-sheet structure. A prion domain determines the infectious properties of a protein by forming the core of the amyloid. We compare the properties of known prion domains and their interactions with the remainder of the protein and with chaperones. Ure2p and Sup35p, two yeast prion proteins, can still form prions when the prion domains are shuffled, indicating a parallel in-register β-sheet structure.

Compositional Determinants of Prion Formation in Yeast

ABSTRACT Numerous prions (infectious proteins) have been identified in yeast that result from the conversion of soluble proteins into β-sheet-rich amyloid-like protein aggregates. Yeast prion formation is driven primarily by amino acid composition. However, yeast prion domains are generally lacking in the bulky hydrophobic residues most strongly associated with amyloid formation and are instead enriched in glutamines and asparagines. Glutamine/asparagine-rich domains are thought to be involved in both disease-related and beneficial amyloid formation. These domains are overrepresented in eukaryotic genomes, but predictive methods have not yet been developed to efficiently distinguish between prion and nonprion glutamine/asparagine-rich domains. We have developed a novel in vivo assay to quantitatively assess how composition affects prion formation. Using our results, we have defined the compositional features that promote prion formation, allowing us to accurately distinguish between glutamine/asparagine-rich domains that can form prion-like aggregates and those that cannot. Additionally, our results explain why traditional amyloid prediction algorithms fail to accurately predict amyloid formation by the glutamine/asparagine-rich yeast prion domains.

De novo design of synthetic prion domains

Prions are important disease agents and epigenetic regulatory elements. Prion formation involves the structural conversion of proteins from a soluble form into an insoluble amyloid form. In many cases, this structural conversion is driven by a glutamine/asparagine (Q/N)-rich prion-forming domain. However, our understanding of the sequence requirements for prion formation and propagation by Q/N-rich domains has been insufficient for accurate prion propensity prediction or prion domain design. By focusing exclusively on amino acid composition, we have developed a prion aggregation prediction algorithm (PAPA), specifically designed to predict prion propensity of Q/N-rich proteins. Here, we show not only that this algorithm is far more effective than traditional amyloid prediction algorithms at predicting prion propensity of Q/N-rich proteins, but remarkably, also that PAPA is capable of rationally designing protein domains that function as prions in vivo.

Amyloids of Shuffled Prion Domains That Form Prions Have a Parallel In-Register β-Sheet Structure

The [URE3] and [PSI (+)] prions of Saccharomyces cerevisiae are self-propagating amyloid forms of Ure2p and Sup35p, respectively. The Q/N-rich N-terminal domains of each protein are necessary and sufficient for the prion properties of these proteins, forming in each case their amyloid cores. Surprisingly, shuffling either prion domain, leaving amino acid content unchanged, does not abrogate the ability of the proteins to become prions. The discovery that the amino acid composition of a polypeptide, not the specific sequence order, determines prion capability seems contrary to the standard folding paradigm that amino acid sequence determines protein fold. The shuffleability of a prion domain further suggests that the beta-sheet structure is of the parallel in-register type, and indeed, the normal Ure2 and Sup35 prion domains have such a structure. We demonstrate that two shuffled Ure2 prion domains capable of being prions form parallel in-register beta-sheet structures, and our data indicate the same conclusion for a single shuffled Sup35 prion domain. This result confirms our inference that shuffleability indicates parallel in-register structure.

Yeast Prions and Human Prion-like Proteins: Sequence Features and Prediction Methods

Prions are self-propagating infectious protein isoforms. A growing number of prions have been identified in yeast, each resulting from the conversion of soluble proteins into an insoluble amyloid form. These yeast prions have served as a powerful model system for studying the causes and consequences of prion aggregation. Remarkably, a number of human proteins containing prion-like domains, defined as domains with compositional similarity to yeast prion domains, have recently been linked to various human degenerative diseases, including amyotrophic lateral sclerosis. This suggests that the lessons learned from yeast prions may help in understanding these human diseases. In this review, we examine what has been learned about the amino acid sequence basis for prion aggregation in yeast, and how this information has been used to develop methods to predict aggregation propensity. We then discuss how this information is being applied to understand human disease, and the challenges involved in applying yeast prediction methods to higher organisms.

The effects of amino acid composition on yeast prion formation and prion domain interactions

Yeast prions provide a powerful model system for examining prion formation and propagation in vivo. Yeast prion formation is driven primarily by amino acid composition, not by primary amino acid sequence. However, although yeast prion domains are consistently glutamine/asparagine-rich, they otherwise vary significantly in their compositions. Therefore, elucidating the exact compositional requirements for yeast prion formation has proven challenging. We have developed an in vivo method that allows for estimation of the prion propensity of each amino acid within the context of a yeast prion domain.1 Using these values, we are able to predict the prion-propensity of various glutamine/asparagine-rich yeast domains. These results provide insight into the basis for yeast prion formation, and may aid in the discovery of additional novel prion domains. Additionally, we examined whether amino acid composition could drive interactions between heterologous glutamine/asparagine-rich proteins.2 Although inefficient interactions between yeast prion domains have previously been observed, we found that one prion protein, Ure2, is able to interact with compositionally similar domains with unprecedented efficiency. This observation, combined with the growing number of yeast prions, suggests that a broad network of interactions between heterologous glutamine/asparagine-rich proteins may affect yeast prion formation.

Distinct Amino Acid Compositional Requirements for Formation and Maintenance of the [PSI+] Prion in Yeast

ABSTRACT Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.