Conserved functions of prion candidates suggest a primeval role of protein self-templating.

Amyloid-based prions have simple structures, a wide phylogenetic distribution, and a plethora of functions in contemporary organisms, suggesting they may be an ancient phenomenon. However, this hypothesis has yet to be addressed with a systematic, computational, and experimental approach. Here we present a framework to help guide future experimental verification of candidate prions with conserved functions to understand their role in the early stages of evolution and potentially in the origins of life. We identified candidate prions in all high-quality proteomes available in UniProt computationally, assessed their phylogenomic distributions, and analyzed candidate-prion functional annotations. Of the 27 980 560 proteins scanned, 228 561 were identified as candidate prions (~0.82%). Among these candidates, there were 84 Gene Ontology (GO) terms conserved across the three domains of life. We found that candidate prions with a possible role in adaptation were particularly well-represented within this group. We discuss unifying features of candidate prions to elucidate the primeval roles of prions and their associated functions. Candidate prions annotated as transcription factors, DNA binding, and kinases are particularly well suited to generating diverse responses to changes in their environment and could allow for adaptation and population expansion into more diverse environments. We hypothesized that a relationship between these functions and candidate prions could be evolutionarily ancient, even if individual prion domains themselves are not evolutionarily conserved. Candidate prions annotated with these universally occurring functions potentially represent the oldest extant prions on Earth and are therefore excellent experimental targets.

Synthetic cells in biomedical applications.

Synthetic cells are engineered vesicles that can mimic one or more salient features of life. These features include directed localization, sense-and-respond behavior, gene expression, metabolism, and high stability. In nanomedicine, many of these features are desirable capabilities of drug delivery vehicles but are difficult to engineer. In this focus article, we discuss where synthetic cells offer unique advantages over nanoparticle and living cell therapies. We review progress in the engineering of the above life-like behaviors and how they are deployed in nanomedicine. Finally, we assess key challenges synthetic cells face before being deployed as drugs and suggest ways to overcome these challenges. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures.

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.

TCF7L2 regulates postmitotic differentiation programmes and excitability patterns in the thalamus.

Neuronal phenotypes are controlled by terminal selector transcription factors in invertebrates, but only a few examples of such regulators have been provided in vertebrates. We hypothesised that TCF7L2 regulates different stages of postmitotic differentiation in the thalamus, and functions as a thalamic terminal selector. To investigate this hypothesis, we used complete and conditional knockouts of Tcf7l2 in mice. The connectivity and clustering of neurons were disrupted in the thalamo-habenular region in Tcf7l2-/- embryos. The expression of subregional thalamic and habenular transcription factors was lost and region-specific cell migration and axon guidance genes were downregulated. In mice with a postnatal Tcf7l2 knockout, the induction of genes that confer thalamic terminal electrophysiological features was impaired. Many of these genes proved to be direct targets of TCF7L2. The role of TCF7L2 in terminal selection was functionally confirmed by impaired firing modes in thalamic neurons in the mutant mice. These data corroborate the existence of master regulators in the vertebrate brain that control stage-specific genetic programmes and regional subroutines, maintain regional transcriptional network during embryonic development, and induce terminal selection postnatally.

Identification of prion protein-derived peptides of potential use in Alzheimer's disease therapy.

Soluble form of the prion protein (PrP) has been previously shown to interact with amyloid-ß (Aß) peptides, suppressing their fibrillization as well as toxicity, which indicates that this protein may play a protective role in Alzheimer's disease (AD). The shortest known PrP fragment retaining all of these properties corresponds to physiologically generated proteolytic polypeptide PrP23-110/111, called N1. Here we have identified two N1-derived synthetic peptides, encompassing residues 23-50 (PrP23-50) and 90-112 (PrP90-112), which bind to Aß1-42 protofibrillar oligomers as well as amyloid fibrils. We found that, akin to N1, the abovementioned synthetic peptides not only reduce the initial rate of Aß fibrillization, but also alter the aggregation pathway of Aß, inhibiting formation of protofibrillar oligomers and facilitating amorphous aggregation. Furthermore, our data show that N1, PrP23-50 and PrP90-112 protect cultured hippocampal neurons from neurotoxic effects of Aß oligomers, preventing oligomers-induced retraction of neurites and loss of cell membrane integrity. The above PrP fragments can also attenuate neuronal intake of Aß. Our results strongly suggest that synthetic peptides such as PrP23-50 and PrP90-112 can be useful in designing a novel class of therapeutics in AD.

Friedreich ataxia is not only a GAA repeats expansion disorder: implications for molecular testing and counselling.

Friedreich ataxia (FRDA) is the most common hereditary ataxia. It is an autosomal recessive disorder caused by mutations of the FXN gene, mainly the biallelic expansion of the (GAA)n repeats in its first intron. Heterozygous expansion/point mutations or deletions are rare; no patients with two point mutations or a point mutation/deletion have been described, suggesting that loss of the FXN gene product, frataxin, is lethal. This is why routine FRDA molecular diagnostics is focused on (GAA)n expansion analysis. Additional tests are considered only in cases of heterozygous expansion carriers and an atypical clinical picture. Analyses of the parent's carrier status, together with diagnostic tests, are performed in rare cases, and, because of that, we may underestimate the frequency of deletions. Even though FXN deletions are characterised as 'exquisitely rare,' we were able to identify one case (2.4 %) of a (GAA)n expansion/exonic deletion in a group of 41 probands. This was a patient with very early onset of disease with rapid progression of gait instability and hypertrophic cardiomyopathy. We compared the patient's clinical data to expansion/deletion carriers available in the literature and suggest that, in clinical practice, the FXN deletion test should be taken into account in patients with early-onset, rapid progressive ataxia and severe scoliosis.

Stabilization of microtubular cytoskeleton protects neurons from toxicity of N-terminal fragment of cytosolic prion protein.

Prion protein (PrP) mislocalized in the cytosol has been presumed to be the toxic entity responsible for the neurodegenerative process in transmissible spongiform encephalopathies (TSE), also called prion diseases. The mechanism underlying the neurotoxicity of cytosolic PrP (cytoPrP) remains, however, unresolved. In this study we analyze toxic effects of the cell-penetrating PrP fragment, PrP1-30--encompassing residues responsible for binding and aggregation of tubulin. We have found that intracellularly localized PrP1-30 disassembles microtubular cytoskeleton of primary neurons, which leads to the loss of neurites and, eventually, necrotic cell death. Accordingly, stabilization of microtubules by taxol reduced deleterious effects of cytosolic PrP1-30. Furthermore, we have demonstrated that decreased phosphorylation level of microtubule-associated proteins (MAPs), which also increases stability of microtubular cytoskeleton, protects neurons from the toxic effects of PrP1-30. CHIR98014 and LiCl--inhibitors of glycogen synthase kinase 3 (GSK-3), a major kinase responsible for phosphorylation of MAPs, inhibited PrP1-30-induced disruption of microtubular cytoskeleton and increased viability of peptide-treated neurons. We have also shown that the N-terminal fragment of cytoPrP may cause the loss of dendritic spines. PrP1-30-induced changes at the level of spines have also been prevented by stabilization of microtubules by taxol as well as LiCl. These observations indicate that the neurotoxicity of cytoPrP is tightly linked to the disruption of microtubular cytoskeleton. Importantly, this study implies that lithium, the commonly used mood stabilizer, may be a promising therapeutic agent in TSE, particularly in case of the disease forms associated with accumulation of cytoPrP.

Prion protein impairs kinesin-driven transport.

Our previous studies have demonstrated that prion protein (PrP) leads to disassembly of microtubular cytoskeleton through binding to tubulin and its oligomerization. Here we found that PrP-treated cells exhibited improper morphology of mitotic spindles. Formation of aberrant spindles may result not only from altered microtubule dynamics - as expected from PrP-induced tubulin oligomerization - but also from impairing the function of molecular motors. Therefore we checked whether binding of PrP to microtubules affected movement generated by Ncd - a kinesin responsible for the proper organization of division spindles. We found that PrP inhibited Ncd-driven transport of microtubules. Most probably, the inhibition of the microtubule movement resulted from PrP-induced changes in the microtubule structure since Ncd-microtubule binding was reduced already at low PrP to tubulin molar ratios. This study suggests another plausible mechanism of PrP cytotoxicity related to the interaction with tubulin, namely impeding microtubule-dependent transport.