PrPC与蛋白质X结合位点可及性的初步探讨

根据PDB提供的PrPC的原子坐标,利用MSMS程序,对PrPC氨基酸残基溶剂可及表面积进行了计算和分析.结果表明:(1) PrPC氨基酸残基可及性具有总体一致性特点;(2) PrPC蛋白质序列中非保守残基与种间屏障有一定关系;(3) 在PrPC向PrP Sc转变过程中,由于蛋白质X的结合,PrPC可能会出现一定的构象变化,这种变化利于PrPC向PrPSc发生转变.     

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.     

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model

The time dependency of the spontaneous aggregation of the fibrillogenic -Amyloid peptide, A^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-A antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 M amyloid peptide. The anti–A antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length A was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the A^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of A^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of A^1–40 in vitro and possibly in vivo.     

Impairment of microtubule system increases alpha-synuclein aggregation and toxicity

The accumulation of fibrillar form of α-synuclein (α-syn) has been implicated in Parkinson’s disease. Here we show that tubulin can stimulate α-syn fibrillization in vitro in different ways depending on its oligomeric status. The physiological significance of tubulin-seeded α-syn fibrillization is demonstrated by using Saccharomyces cerevisiae as a model system. Perturbation of microtubule system either by treating benomyl that inhibits microtubule assembly or by deleting genes involved in microtubule biogenesis, stimulates α-syn aggregation and toxicity. These results suggest that impairment of the microtubule system may act as a risk factor deteriorating the α-syn-mediated neurodegeneration by increasing the chance of tubulin-seeded α-syn aggregation.     

Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection

Diseases such as type 2 diabetes, Alzheimer's and Parkinson's are associated with the formation of amyloid. The transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease, are believed to result from infectious forms of amyloid proteins termed prions. The ability of amyloid to initiate spontaneously and in the case of prions, to transfer successfully from one host to another, has been hard to fully rationalize. In this paper we use a mathematical model to explore the idea that it might be a combination of the presence of the prion/amyloid form and a change in the state of the host that allows the amyloid/prion to successfully initiate and propagate itself. We raise the intriguing possibility that potentially infectious amyloid may lie dormant in an apparently healthy individual awaiting a change in the state of the host or transmittal to a new more susceptible host. On this basis we make an analogy between prion/amyloid disease development and the two-hit model of cancer progression. We additionally raise the possibility that infectious amyloid strains may be characterized by a size distribution of length or radius.     

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein

[PSI⁺] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI⁺] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI⁺] prions typically affect viability only modestly, so [PSI⁺] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI⁺] propagation, yet [PSI⁺] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI⁺] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI⁺] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI⁺] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI⁺] prions.     

Phosphorylation of the FUS low‐complexity domain disrupts phase separation,aggregation, and toxicity

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.     

Small heat shock proteins differentially affect Abeta aggregation and toxicity

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.     

杀虫剂对烟粉虱成虫毒力的微量筛选方法

采用直径为18mm的指形管代替常规浸叶法中的培养皿,研究不同寄主植物叶片和琼脂浓度对烟粉虱Bemisia tabaci(Gennadius)成虫自然死亡的影响,并用吡虫啉和啶虫脒为供试药剂对烟粉虱成虫毒力的筛选方法进行比较。结果表明:选用棉花叶片和14g.L-1的琼脂时,指形管内烟粉虱成虫72h自然死亡率最低,只有(5.4±1.5)%;采用此方法就吡虫啉和啶虫脒对烟粉虱成虫的毒力进行测定,测定结果重复性较好,节省药剂用量,无需对烟粉虱成虫麻醉,操作简便,达到微量活体筛选的目标。     

Influence of the N-terminal domain on the aggregation properties of the prion protein

Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrP(C)) into an infectious form denoted PrP(Sc). The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrP(Sc) is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein because it contains a largely structured, alpha-helical, C-terminal region in addition to an N terminus that is unstructured in the absence of metal ion binding. Herein, the in vitro aggregation of a truncated portion of the prion protein (PrP 90-231) and a full-length version (PrP 23-231) were compared. In each case, concentration-dependent aggregation was analyzed to discern whether it proceeds by a nucleation-dependent pathway. Both protein constructs appear to aggregate via a nucleated polymerization with a small nucleus size, yet the later steps differ. The full-length protein forms larger aggregates than the truncated protein, indicating that the N terminus may mediate higher-order aggregation processes. In addition, the N terminus has an influence on the assembly state of PrP before aggregation begins, causing the full-length protein to adopt several oligomeric forms in a neutral pH buffer. Our results emphasize the importance of studying the full-length protein in addition to the truncated forms for in vitro aggregation studies in order to make valid hypotheses about the mechanisms of prion aggregation and the distribution of aggregates in vivo.     

In vivo evidence of CHIP up-regulation attenuating tau aggregation

The carboxyl terminus of heat-shock cognate (Hsc)70-interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat-shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimer's disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIP's role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl-insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non-transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl-insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality-control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies.     

Yeast prions are useful for studying protein chaperones and protein quality control

Abstract

Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell's protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.     

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem

Abstract

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.     

The same but different: the role of Hsp70 in heat shock response and prion propagation

ABSTRACT

The Hsp70 chaperone machinery is a key component of the heat-shock response and a modulator of prion propagation in yeast. A major factor in optimizing Hsp70 function is the highly coordinated activities of the nucleotide-binding and substrate-binding domains of the protein. Hsp70 inter-domain communication occurs through a bidirectional allosteric interaction network between the two domains. Recent findings identified the β6/β7 region of the substrate-binding domain as playing a critical role in optimizing Hsp70 function in both the stress response and prion propagation and highlighted the allosteric interaction interface between the domains. Importantly, while functional changes in Hsp70 can result in phenotypic consequences for both the stress response and prion propagation, there can be significant differences in the levels of phenotypic impact that such changes illicit.     

Yeast prions are useful for studying protein chaperones and protein quality control

Protein chaperones help proteins adopt and maintain native conformations and play vital roles in cellular processes where proteins are partially folded. They comprise a major part of the cellular protein quality control system that protects the integrity of the proteome. Many disorders are caused when proteins misfold despite this protection. Yeast prions are fibrous amyloid aggregates of misfolded proteins. The normal action of chaperones on yeast prions breaks the fibers into pieces, which results in prion replication. Because this process is necessary for propagation of yeast prions, even small differences in activity of many chaperones noticeably affect prion phenotypes. Several other factors involved in protein processing also influence formation, propagation or elimination of prions in yeast. Thus, in much the same way that the dependency of viruses on cellular functions has allowed us to learn much about cell biology, the dependency of yeast prions on chaperones presents a unique and sensitive way to monitor the functions and interactions of many components of the cell''s protein quality control system. Our recent work illustrates the utility of this system for identifying and defining chaperone machinery interactions.     

Prediction of the aggregation propensity of proteins from the primary sequence: aggregation properties of proteomes

In the cell, protein folding into stable globular conformations is in competition with aggregation into non-functional and usually toxic structures, since the biophysical properties that promote folding also tend to favor intermolecular contacts, leading to the formation of β-sheet-enriched insoluble assemblies. The formation of protein deposits is linked to at least 20 different human disorders, ranging from dementia to diabetes. Furthermore, protein deposition inside cells represents a major obstacle for the biotechnological production of polypeptides. Importantly, the aggregation behavior of polypeptides appears to be strongly influenced by the intrinsic properties encoded in their sequences and specifically by the presence of selective short regions with high aggregation propensity. This allows computational methods to be used to analyze the aggregation properties of proteins without the previous requirement for structural information. Applications range from the identification of individual amyloidogenic regions in disease-linked polypeptides to the analysis of the aggregation properties of complete proteomes. Herein, we review these theoretical approaches and illustrate how they have become important and useful tools in understanding the molecular mechanisms underlying protein aggregation.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

Al (III) metal augment thermal aggregation and fibrillation in protein: Role of metal toxicity in neurological diseases

Protein fibrillation is a leading cause of innumerable neurodegenerative diseases. The exact underlying mechanism associated with the formation of fibrils is yet to be known. Recently, the role of metal ions resulting into fibrillation of proteins has gained attention of the scientific community. In this piece of work, we have investigated the effect of the aluminum (Al) metal ion on the kinetics of aggregation of bovine serum albumin (BSA) protein under physiological conditions by employing several biophysical and microscopic techniques. Quenching of tryptophan fluorescence was observed along with 9 nm blue shift, demonstrating BSA becomes more hydrophobic during unfolding pathway of thermal denaturation. Moreover, ANS (8-Anilino-1-naphthalene sulfonic acid) binding shows quenching in fluorescence intensity with increasing time of incubation at 65 °C, suggesting unfolding leading to the disruption of hydrophobic patches in BSA. Besides, Thioflavin T intensity indicated a significant acceleration in BSA fibrillation at a ratio of 1:1 and 1:2 of BSA and Al (III) metal ion respectively. In addition, circular dichroism (CD) spectroscopy study revealed the transition of BSA from α-helical conformation to the β-sheet rich structure. Molecular docking analysis demonstrated significant binding affinity (−1.2 kcal/mol) of Al (III) with BSA involving Phe501, Phe506, Val575, Thr578, Gln579, Leu531 residues. Transmission electron microscopy (TEM) reaffirm augmentation of thermal-induced BSA fibril formation in the presence of Al (III) metal ions. This study highlights the metal chelating potency as the possible therapeutic target for neurological diseases.     

Beta-amyloid-derived pentapeptide RIIGLa inhibits Abeta(1-42) aggregation and toxicity

Pr-IIGL(a), a derivative of the tetrapeptide beta-amyloid 31-34 (Abeta(31-34)), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Abeta(1-42). For an understanding of this phenomenon, a new pentapeptide, RIIGL(a) was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL(a) forms fibrillar aggregates, whereas RIIGL(a) does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL(a) acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL(a) does not display inherent toxicity. RIIGL(a) co-incubated with Abeta(1-42) inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Abeta(1-42). These results indicate that RIIGL(a) is an effective inhibitor of both the aggregation and the toxic effects of Abeta(1-42) and can serve as a lead compound for the design of novel neuroprotective peptidomimetics.     

Stability of single sheet GNNQQNY aggregates analyzed by replica exchange molecular dynamics: antiparallel versus parallel association

Protein and peptide aggregation into amyloid plaques is associated with a large variety of neurodegenerative diseases. The definition of the molecular bases of these pathologies is hampered by the transient nature of pre-fibrillar small-oligomers that are considered the toxic species. The ability of the peptide GNNQQNY to form amyloid-like structures makes it a good model to investigate the complex processes involved into amyloid fiber formation. By employing full atomistic replica exchange molecular dynamics simulations, we constructed the free energy surface of small assemblies of GNNQQNY to gain novel insights into the fiber formation process. The calculations suggest that the peptide exhibits a remarkable tendency to form both parallel and antiparallel β-sheets. The data show that GNNQQNY preference for parallel or antiparallel β-sheets is governed by a subtle balance of factors including assemblies’ size, sidechain-sidechain interactions and pH. The samplings analysis provides a rationale to the observed trends.