全长朊粒蛋白淀粉样纤维引发细胞毒性的机制研究

目的 朊病毒病(prion disease)是一类由朊粒蛋白(PrP)发生错误折叠、聚集形成致病性的PrP^Sc导致的具有高致死率的神经退行性疾病。本文在细胞和动物水平开展了PrP纤维诱导内源PrP聚集和毒性机制的研究。方法 通过超速离心结合蛋白质免疫印迹实验检测PrP聚集；通过氧化压力实验，使用Annexin V-FITC/PI双染检测细胞凋亡；运用细胞超薄切片技术检测细胞线粒体形态；在动物水平，分离新生小鼠的前额叶，进行横断切片培养，在脑片上接种PrP纤维。结果 PrP纤维种子可以诱导内源PrP聚集，PrP纤维可以诱导细胞内氧化压力升高和细胞凋亡，PrP纤维可以引起线粒体损伤，PrP纤维可以诱导小鼠前额叶内源PrP聚集。结论 本文在细胞和动物水平证实体外组装的PrP淀粉样纤维具有细胞毒性和潜在的感染性。     

Disease-associated protein seeding suggests a dissociation between misfolded protein accumulation and neurodegeneration in prion disease

Chronic neurodegenerative diseases, such as prion diseases or Alzheimer's disease, are associated with progressive accumulation of host proteins which misfold and aggregate. Neurodegeneration is restricted to specific neuronal populations which show clear accumulation of misfolded proteins, whilst neighbouring neurons remain unaffected. Such data raise interesting questions about the vulnerability of specific neuronal populations to neurodegeneration and much research has concentrated only on the mechanisms of neurodegeneration in afflicted neuronal populations. An alternative, undervalued and almost completely unstudied question however is how and why neuronal populations are resilient to neurodegeneration. One potential answer is unaffected regions do not accumulate misfolded proteins, thus mechanisms of neurodegeneration do not become activated. In this perspectives, we discuss novel data from our laboratories which demonstrate that misfolded proteins do accumulate in regions of the brain which do not show evidence of neurodegeneration and further evidence that microglial responses may define the severity of neurodegeneration.     

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.     

The aminoglycoside G418 hinders de novo prion infection in cultured cells

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.     

A molecular dynamics approach to the structural characterization of amyloid aggregation

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.     

The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein

In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into β‐sheet rich amyloid‐like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin‐T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108–143 of the human prion protein [humPrP (108–143)]—the evolutionary most conserved part of the prion protein ‐ residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108–143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108–143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles.     

Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer＇s disease, Parkinson＇s disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggre- gation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute signifi- cantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both aU-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders.     

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN⁺], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.     

Binding mechanism of patulin to heat‐treated yeast cell

Aims

This study aims to assess the removal mechanism of patulin using heat‐treated Saccharomyces cerevisiae cells and identify the role of different cell wall components in the binding process.

Methods and Results

In order to understand the binding mechanism, viable cells, heat‐treated cells, cell wall and intracellular extract were performed to assess their ability to remove patulin. Additionally, the effects of chemical and enzymatic treatments of yeast on the binding ability were tested. The results showed that there was no significant difference between viable (53·28%) and heat‐treated yeast cells (51·71%) in patulin binding. In addition, the cell wall fraction decreased patulin by 35·05%, and the cell extract nearly failed to bind patulin. Treatments with protease E, methanol, formaldehyde, periodate or urea significantly decreased (P < 0·05) the ability of heat‐treated cells to remove patulin. Fourier transform infrared (FTIR) analysis indicated that more functional groups were involved in the binding process of heat‐treated cells.

Conclusions

Polysaccharides and protein are important components of yeast cell wall involved in patulin removal. In addition, hydrophobic interactions play a major role in binding processes.

Significance and Impact of the Study

Heat‐treated S. cerevisiae cells could be used to control patulin contamination in the apple juice industry. Also, our results proof that the patulin removal process is based mainly on the adsorption not degradation.     

Misfolding leads the way to unraveling signaling pathways in the pathophysiology of prion diseases

A misfolded version of the prion protein represents an essential component in the pathophysiology of fatal neurodegenerative prion diseases, which affect humans and animals alike. They may be of sporadic origin, acquired through exogenous introduction of infectious misfolded prion protein, or caused by genetic alterations in the prion protein coding gene. We have recently described a novel pathway linking retention of mutant prion protein in the early secretory pathway to activation p38-MAPK and a neurodegenerative phenotype in transgenic mice. Here we review the consequences that mutations in prion protein have on intracellular transport and stress responses focusing on protein quality control. We also discuss the neurotoxic signaling elicited by the accumulation of mutant prion protein in the endoplasmic reticulum and the Golgi apparatus. Improved knowledge about these processes will help us to better understand complex pathogenesis of prion diseases, a prerequisite for therapeutic strategies.     

A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration

Most neurodegenerative diseases such as Alzheimer''s disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p‐bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.     

Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.     

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.     

Mutagenic exploration of the cross-seeding and fibrillation propensity of Alzheimer's beta-amyloid peptide variants

Amyloid formation is a nucleation-dependent process that is accelerated dramatically in vivo and in vitro upon addition of appropriate fibril seeds. A potent species barrier can be effective in this reaction if donor and recipient come from different biological species. This species barrier is thought to reflect differences in the amino acid sequence between seed and target polypeptide. Here we present an in vitro mutagenic cross-seeding analysis of Alzheimer's Abeta(1-40) peptide in which we mapped out the effect of systematically varied amino acid replacements on the propensity of seed-dependent amyloid fibril formation. We find that the susceptibility of different peptides toward cross-seeding relates to the intrinsic aggregation propensity of the respective polypeptide chain and, therefore, to properties such as beta-sheet propensity and hydrophobicity. These data imply that the seed-dependent formation of amyloid-like fibrils is affected by the intrinsic properties of the polypeptide chain in a manner that is similar to what has been described previously for aggregation reactions in general. Hence, the nucleus acts in this case as a catalyst that promotes the fibrillation of different polypeptide chains according to their intrinsic structural predilection.     

A molecular dynamics study of acylphosphatase in aggregation-promoting conditions: the influence of trifluoroethanol/water solvent

The 98-residue protein acylphosphatase exhibits a high propensity for aggregation under certain conditions. Aggregates formed from wild-type acylphosphatase in the presence of 2,2,2-trifluoroethanol and from highly destabilized mutants are essentially identical in structure. Furthermore, it has been shown by mutational studies that different regions of the protein are important for aggregation and folding. In the present molecular dynamics study, we compare the behavior of the protein in aqueous solution and in a 25% (v/v) 2,2,2-trifluoroethanol/water environment mimicking the experimental conditions. The 2,2,2-trifluoroethanol surrounding affects the structure of the protein mostly in the regions important for aggregation, in good agreement with experimental data. This suggests that the early step of (partly) unfolding, which precedes the aggregation process, has been observed.     

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta transition mechanism revealed by molecular dynamics simulations

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid-related disorders. In the present study the transition from ideal alpha-helical to beta-hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well-known amyloidogenic peptides: the H1 peptide from prion protein and the Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side-chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2-trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation.     

Architecture of the yeast Elongator complex

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1‐6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub‐complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two‐lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.     

Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.