Transition of the prion protein from a structured cellular form (PrPC) to the infectious scrapie agent (PrPSc)

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrP^C) to a pathological isoform called “prion protein scrapie” (PrP^Sc). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X‐ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three‐dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure‐based drug discovery endeavors.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

The story of stolen chaperones

Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller “seeds.” Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI⁺] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI⁺] aggregates to enlarge. This is incompatible with a previously proposed “capping” model where the overexpressed Q/N-rich protein poisons, or “caps,” the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI⁺] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI⁺] aggregates in a way that blocks their shearing.     

Allelic variants of hereditary prions: The bimodularity principle

Modern biology requires modern genetic concepts equally valid for all discovered mechanisms of inheritance, either “canonical” (mediated by DNA sequences) or epigenetic. Applying basic genetic terms such as “gene” and “allele” to protein hereditary factors is one of the necessary steps toward these concepts. The basic idea that different variants of the same prion protein can be considered as alleles has been previously proposed by Chernoff and Tuite. In this paper, the notion of prion allele is further developed. We propose the idea that any prion allele is a bimodular hereditary system that depends on a certain DNA sequence (DNA determinant) and a certain epigenetic mark (epigenetic determinant). Alteration of any of these 2 determinants may lead to establishment of a new prion allele. The bimodularity principle is valid not only for hereditary prions; it seems to be universal for any epigenetic hereditary factor.     

Is tau ready for admission to the prion club?

Aggregation-prone proteins associated with neurodegenerative disease, such as α synuclein and β amyloid, now appear to share key prion-like features with mammalian prion protein, such as the ability to recruit normal proteins to aggregates and to translocate between neurons. These features may shed light on the genesis of stereotyped lesion development patterns in conditions such as Alzheimer disease and Lewy Body dementia. We discuss the qualifications of tau protein as a possible “prionoid” mediator of lesion spread based on recent characterizations of the secretion, uptake and transneuronal transfer of human tau isoforms in a variety of tauopathy models, and in human patients. In particular, we consider (1) the possibility that prionoid behavior of misprocessed tau in neurodegenerative disease may involve other aggregation-prone proteins, including PrP itself, and (2) whether “prionlike” tau lesion propagation might include mechanisms other than protein-protein templating.     

Comparative profiling of highly enriched 22L and Chandler mouse scrapie prion protein preparations

Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrP^Sc, appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP^Sc‐associated proteins which help to determine TSE strain‐specific disease phenotypes. We enriched PrP^Sc from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC‐MS/MS. These samples were compared with “mock” PrP^Sc preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrP^Sc samples from 22L and Chandler TSE strains suggest that the non‐PrP^Sc protein components found in standard enrichment protocols are not strain specific.     

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity

In Alzheimer's disease and tauopathies, tau protein aggregates into neurofibrillary tangles that progressively spread to synaptically connected brain regions. A prion‐like mechanism has been suggested: misfolded tau propagating through the brain seeds neurotoxic aggregation of soluble tau in recipient neurons. We use transgenic mice and viral tau expression to test the hypotheses that trans‐synaptic tau propagation, aggregation, and toxicity rely on the presence of endogenous soluble tau. Surprisingly, mice expressing human P301Ltau in the entorhinal cortex showed equivalent tau propagation and accumulation in recipient neurons even in the absence of endogenous tau. We then tested whether the lack of endogenous tau protects against misfolded tau aggregation and toxicity, a second prion model paradigm for tau, using P301Ltau‐overexpressing mice with severe tangle pathology and neurodegeneration. Crossed onto tau‐null background, these mice had similar tangle numbers but were protected against neurotoxicity. Therefore, misfolded tau can propagate across neural systems without requisite templated misfolding, but the absence of endogenous tau markedly blunts toxicity. These results show that tau does not strictly classify as a prion protein.     

Potential mechanisms and implications for the formation of tau oligomeric strains

The culmination of many years of increasing research into the toxicity of tau aggregation in neurodegenerative disease has led to the consensus that soluble, oligomeric forms of tau are likely the most toxic entities in disease. While tauopathies overlap in the presence of tau pathology, each disease has a unique combination of symptoms and pathological features; however, most study into tau has grouped tau oligomers and studied them as a homogenous population. Established evidence from the prion field combined with the most recent tau and amyloidogenic protein research suggests that tau is a prion-like protein, capable of seeding the spread of pathology throughout the brain. Thus, it is likely that tau may also form prion-like strains or diverse conformational structures that may differ by disease and underlie some of the differences in symptoms and pathology in neurodegenerative tauopathies. The development of techniques and new technology for the detection of tau oligomeric strains may, therefore, lead to more efficacious diagnostic and treatment strategies for neurodegenerative disease.     

Alzheimer disease

Alzheimer disease (AD) has traditionally been thought to involve the misfolding and aggregation of two different factors that contribute in parallel to pathogenesis: amyloid-β (Aβ) peptides, which represent proteolytic fragments of the transmembrane amyloid precursor protein, and tau, which normally functions as a neuronally enriched, microtubule-associated protein that predominantly accumulates in axons. Recent evidence has challenged this model, however, by revealing numerous functional interactions between Aβ and tau in the context of pathogenic mechanisms for AD. Moreover, the propagation of toxic, misfolded Aβ and tau bears a striking resemblance to the propagation of toxic, misfolded forms of the canonical prion protein, PrP, and misfolded Aβ has been shown to induce tau misfolding in vitro through direct, intermolecular interaction. In this review we discuss evidence for the prion-like properties of both Aβ and tau individually, as well as the intriguing possibility that misfolded Aβ acts as a template for tau misfolding in vivo.     

Tau融合蛋白及其缺失突变体与朊蛋白的体外作用分析

在部分朊病毒病（prion diseases）中,高度磷酸化的微管相关蛋白tau与朊蛋白(prion protein,PrP)发生共定位,tau蛋白可能在朊病毒病的病理机制中有重要作用. 本室已经证明二者可以发生分子间相互作用,本文进一步分析了tau蛋白与prion的体外相互作用及作用位点. 利用RT-PCR方法从人源细胞系SHSY5Y cDNA中扩增出微管相关蛋白tau全长cDNA序列,克隆至质粒pGEX-2T载体,在大肠杆菌中诱导表达融合蛋白GST-tau. 利用GST pull-down及免疫共沉淀方法检测全长tau蛋白与PrP23-231的分子间相互作用. 进一步表达tau 蛋白的各种缺失突变体,确定tau蛋白与PrP蛋白的相互作用位点. 结果表明,所表达的全长tau蛋白及各种缺失突变体均为可溶性蛋白,Western印迹结果显示,各种蛋白均能很好的被tau蛋白单抗识别. GST pull-down和免疫共沉淀实验均显示,原核表达的全长tau蛋白可与全长的PrP蛋白在体外发生相互作用,并确定相互作用位点位于tau蛋白的N端序列及中段的重复区. 上述结果为研究tau蛋白与PrP的相互作用在朊病毒病的发病机制中的意义提供了一定的理论基础.     

Mercury(II) promotes the in vitro aggregation of tau fragment corresponding to the second repeat of microtubule‐binding domain: Coordination and conformational transition

The loss of metal homeostasis and the toxic effect of metal ion are important events in neurodegenerative and age‐related diseases, such as Alzheimer's disease (AD). For the first time, we investigated the impacts of mercury(II) ions on the folding and aggregation of Alzheimer's tau fragment R2 (residues 275‐305: VQIIN KKLDL SNVQS KCGSK DNIKH VPGGGS), corresponding to the second repeat unit of the microtubule‐binding domain, which was believed to be pivotal to the biochemical properties of full tau protein. By ThS fluorescence assay and electron microscopy, we found that mercury(II) dramatically promoted heparin‐induced aggregation of R2 at an optimum molar ratio of 1: 2 (metal: protein), and the resulting R2 filaments became smaller. Isothermal titration calorimetry (ITC) experiment revealed that the strong coordination of mercury(II) with R2 was an enthalpy‐controlled, entropy‐decreased thermodynamic process. The exceptionally large magnitude of heat release (ΔH₁ = ?34.8 Kcal mol^?1) suggested that the most possible coordinating site on the R2 peptide chain was the thiol group of cysteine residue (Cys291), and this was further confirmed by a control experiment using Cys291 mutated R2. Circular dichroism spectrum demonstrated that this peptide underwent a significant conformational change from random coil to β‐turn structure upon its binding to mercury(II) ion. This study was undertaken to better understand the mechanism of tau aggregation, and evaluate the possible role of mercury(II) in the pathogenesis of AD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1100–1107, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The 12‐15‐lipoxygenase is a modulator of Alzheimer's‐related tau pathology in vivo

12/15‐lipoxygenase (12‐15LO) is a lipid‐peroxidizing enzyme widely expressed in the central nervous system where it has been involved in the neurobiology of Alzheimer's disease (AD) because it modulates amyloid beta (Aβ) and APP processing. However, its biological effect on tau protein is unknown. We investigated the effect of 12‐15LO on tau levels and metabolism in vivo and in vitro and the mechanism involved by using genetic and pharmacologic approaches. While no significant differences were observed in the levels of total tau for both groups, compared with controls, Tg2576 mice overexpressing 12‐15LO had elevated levels of phosphorylated tau at two specific epitopes, Ser 202/Thr 205 and Ser 396. In vitro and in vivo studies show that 12‐15LO modulates tau metabolism specifically via the cdk5 kinase pathway. Associated with these changes were biochemical markers of synaptic pathology. Finally, 12‐15LO‐dependent alteration of tau metabolism was independent from an effect on Aβ. Our findings reveal a novel pathway by which 12‐15LO modulates endogenous tau metabolism making this protein an appealing pharmacologic target for treatment of AD and related tauopathies.     

Unusual semi‐extractability as a hallmark of nuclear body‐associated architectural noncoding RNAs

NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20‐fold (a property we term “semi‐extractability”), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi‐extractability. A comparison of RNA‐seq data from needle‐sheared and control samples revealed the existence of multiple semi‐extractable RNAs, many of which were localized in subnuclear granule‐like structures. The semi‐extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion‐like domain of the RNA‐binding protein FUS. This observation suggests that tenacious RNA–protein and protein–protein interactions, which drive nuclear body formation, are responsible for semi‐extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.     

A role for the proteasome in the turnover of Sup35p and in [PSI+] prion propagation

Yeast prions are superb models for understanding the mechanisms of self‐perpetuating protein aggregates formation. [PSI⁺] stands among the most documented yeast prions and results from self‐assembly of the translation termination factor Sup35p into protein fibrils. A plethora of cellular factors were shown to affect [PSI⁺] formation and propagation. Clearance of Sup35p prion particles is however poorly understood and documented. Here, we investigated the role of the proteasome in the degradation of Sup35p and in [PSI⁺] prion propagation. We found that cells lacking the RPN4 gene, which have reduced intracellular proteasome pools, accumulated Sup35p and have defects in [PSI⁺] formation and propagation. Sup35p is degraded in vitro by the 26S and 20S proteasomes in a ubiquitin‐independent manner, generating an array of amyloidogenic peptides derived from its prion‐domain. We also demonstrate the formation of a proteasome‐resistant fragment spanning residues 83–685 which is devoid of the prion‐domain that is essential for [PSI⁺] propagation. Most important was the finding that the 26S and 20S proteasomes degrade Sup35p fibrils in vitro and abolish their infectivity. Our results point to an overlooked role of the proteasome in clearing toxic protein aggregates, and have important implications for a better understanding of the life cycle of infectious protein assemblies.     

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP^Sc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.     

Cover Picture: Biotechnology Journal 8/2017

Thaw what is frozen: it is generally accepted that the initial genetic code evolved from an ambiguous to a well‐defined (”frozen“) code with the repertoire of 20 (+2) canonical amino acids. If code is perfectly optimized by evolution, is it then possible to change it experimentally? If so, what are the barriers to overcome? Kubyshkin and Budisa have tried to answer this question by building a model that predicts ”entry points“ for invading the code with noncanonic amino acids. These experiments should lead to a chemical alienation of cells which represent an important step towards the creation of artificial life. The cover is prepared by Vladimir Kubyshkin and Nediljko Budisa authors of the article ”Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?” ( https://doi.org/10.1002/biot.201600097 ).     

Molecular dynamics simulations of early steps in RNA‐mediated conversion of prions

The rate‐limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis‐folded state in which the protein not only forms cell‐toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA‐mediated mis‐folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.