Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, TTR related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects, and particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

Highlights d hnRNPDL requires both N-and C-terminal IDRs to phase separate d The absence of N-terminal IDR facilitates aggregation d The unique combination of IDRs in each isoform determines its cellular behavior d D378N/H mutations accelerate hnRNPDL aggregation and compromise its solubility

Amyloids consist of repetitions of a specific polypeptide chain in a regular cross-β-sheet conformation. Amyloid propensity is largely determined by the protein sequence, the aggregation process being nucleated by specific and short segments. Prions are special amyloids that become self-perpetuating after aggregation. Prions are responsible for neuropathology in mammals, but they can also be functional, as in yeast prions. The conversion of these last proteins to the prion state is driven by prion forming domains (PFDs), which are generally large, intrinsically disordered, enriched in glutamines/asparagines and depleted in hydrophobic residues. The self-assembly of PFDs has been thought to rely mostly on their particular amino acid composition, rather than on their sequence. Instead, we have recently proposed that specific amyloid-prone sequences within PFDs might be key to their prion behaviour. Here, we demonstrate experimentally the existence of these amyloid stretches inside the PFDs of the canonical Sup35, Swi1, Mot3 and Ure2 prions. These sequences self-assemble efficiently into highly ordered amyloid fibrils, that are functionally competent, being able to promote the PFD amyloid conversion in vitro and in vivo. Computational analyses indicate that these kind of amyloid stretches may act as typical nucleating signals in a number of different prion domains.

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A disordered to β-sheet transition was thought to drive the functional switch of Q/N-rich prions, similar to pathogenic amyloids. However, recent evidence indicates a critical role for coiled-coil (CC) regions within yeast prion domains in amyloid formation. We show that many human prion-like domains (PrLDs) contain CC regions that overlap with polyQ tracts. Most of the proteins bearing these domains are transcriptional coactivators, including the Mediator complex subunit 15 (MED15) involved in bridging enhancers and promoters. We demonstrate that the human MED15-PrLD forms homodimers in solution sustained by CC interactions and that it is this CC fold that mediates the transition towards a β-sheet amyloid state, its chemical or genetic disruption abolishing aggregation. As in functional yeast prions, a GFP globular domain adjacent to MED15-PrLD retains its structural integrity in the amyloid state. Expression of MED15-PrLD in human cells promotes the formation of cytoplasmic and perinuclear inclusions, kidnapping endogenous full-length MED15 to these aggregates in a prion-like manner. The prion-like properties of MED15 are conserved, suggesting novel mechanisms for the function and malfunction of this transcription coactivator.

The aberrant or misfolded forms of the prion protein have been described as the causative agents of rare transmissible spongiform encephalopathies. In addition, proteins associated with frequently occurring neurodegenerative disorders, such as Alzheimer's and Parkinson's, are shown to share prion-like properties and to spread the disease in the brain. Areas covered: Interest in the prion phenomenon has crystallized in a series of computational methods aimed at uncovering prion-like proteins at the proteome level. These programs rely on the identification of sequence signatures similar to those of yeast prions, whose structural conversion is driven by specific domains enriched in glutamine/asparagine residues. A myriad of prion-like candidates, similar to those in yeast, are predicted to exist in organisms across all kingdoms of life. We review here the role of prions, prionoids and prion-like proteins in health and disease, with a special focus on the algorithms and databases developed for their prediction and classification. Expert commentary: Computational approaches provide novel insights into prion-like protein functions, their regulation and their role in disease.

Prion-like behaviour is attracting much attention due to the growing evidences that amyloid-like self-assembly may reach beyond neurodegeneration and be a conserved functional mechanism. The best characterized functional prions correspond to a subset of yeast proteins involved in translation or transcription. Their conformational promiscuity is encoded in Prion Forming Domains (PFDs), usually long and intrinsically disordered protein segments of low complexity. The compositional bias of these regions seems to be important for the transition between soluble and amyloid-like states. We have proposed that the presence of cryptic soft amyloid cores embedded in yeast PFDs can also be important for their assembly and demonstrated their existence and self-propagating abilities. Here, we used an orthogonal approach in the search of human domains that share yeast PFDs compositional bias and exhibit a predicted nucleating core, identifying 535 prion-like candidates. We selected seven proteins involved in transcriptional or translational regulation and associated to disease to characterize the properties of their amyloid cores. All of them self-assemble spontaneously into amyloid-like structures able to propagate their polymeric state. This provides support for the presence of short sequences able to trigger conformational conversion in prion-like human proteins, potentially regulating their functionality.

BackgroundAround 1% of human proteins are predicted to contain a disordered and low complexity prion-like domain (PrLD). Mutations in PrLDs have been shown promote a transition towards an aggregation-prone state in several diseases.ResultsRecently, we have shown that an algorithm that considers the effects of mutations on PrLDs composition, as well as on localized amyloid propensity can predict the impact of these amino acid changes on protein intracellular aggregation. In this application note, we implement this concept into the AMYCO web server, a refined algorithm that forecasts the influence of amino acid changes in prion-like proteins aggregation propensity better than state-of-the-art predictors.ConclusionsThe AMYCO web server allows for a fast and automated evaluation of the effect of mutations on the aggregation properties of prion-like proteins. This might uncover novel disease-linked amino acid changes in the sequences of human prion-like proteins. Additionally, it can find application in the in silico design of synthetic prion-like proteins with tuned aggregation propensities for different purposes. AMYCO does not require previous registration and is freely available to all users at: http://bioinf.uab.cat/amyco/.Electronic supplementary materialThe online version of this article (10.1186/s12859-019-2601-3) contains supplementary material, which is available to authorized users.