Ischemic preconditioning induces chaperone hsp70 expression and inhibits protein aggregation in the CA1 neurons of rats

Objective To investigate the effect of ischemic preconditioning on chaperone hsp70 expression and protein aggregation in the CA1 neurons of rats, and to further explore its potential neuroprotective mechanism. Methods Two-vesseloccluded transient global ischemia rat model was used. The rats were divided into sublethal 3-min ischemia group, lethal 10- min ischemia group and ischemic preconditioning group. Neuronal death in the CA1 region was observed by hematoxylineosin staining, and number of live neurons was assessed by cell counting under a light microscope. Immunochemistry and laser scanning confocal microscopy were used to observe the distribution of chaperone hsp70 in the CA1 neurons. Differential centrifuge was used to isolate cytosol, nucleus and protein aggregates fractions. Western blot was used to analyze the quantitative alterations of protein aggregates and inducible chaperone hsp70 in cellular fractions and in protein aggregates under different ischemic conditions. Results Histological examination showed that ischemic preconditioning significantly reduced delayed neuronal death in the hippocampus CA1 region （P 〈 0.01 vs 10-min ischemia group）. Sublethal ischemic preconditioning induced chaperone hsp70 expression in the CA1 neurons after 24 h reperfusion following 10-min ischemia. Induced-hsp70 combined with the abnormal proteins produced during the secondary lethal 10-min ischemia and inhibited the formation of cytotoxic protein aggregates（P〈0.01 vs 10-min ischemia group）.Conelusion Ischemic preconditioning induced chaperone hsp70 expression and inhibited protein aggregates formation in the CA1 neurons when suffered secondary lethal ischemia, which may protect neurons from death.

Pyropia haitanensis polysaccharide extends lifespan by inhibiting protein aggregation in Caenorhabditis elegans

Pyropia haitanensis polysaccharide(LP)have been found for having many excellent functions such as anti-aging.Using Caenorhabditis elegans models,we evaluated the anti-aging activity of LP by observing the lifespan,reproduction,pharyngeal pumping,stress response,quantitative fluorescence of polyglutamic acid,and nuclear localization of DAF-16 of worms.The results reveal that LP could extend the adult lifespan of wild-type and polyQ nematodes,indicating a connection of its anti-aging benefit with the toxicity-suppressing effect.The number of polyglutamic acid aggregates in high concentration groups decreased by 24.39%(P<0.05)to the control.The high-dose group strongly induced DAF-16 nuclear translocation over intermediate and cytosolic localizations compared with the control(P<0.001).Therefore,we believe that LP could extend the lifespan and reduce the protein aggregation in C.elegans through nuclear DAF-16∷GFP expression.

Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity,recapitulating key features of FUS proteinopathy

Mutations in the fused in sarcoma/translocated in liposarcoma(FUS/TLS)gene have been associated with amyotrophic lateral sclerosis(ALS).FUS-positive neuropathology is reported in a range of neurodegenerative diseases,including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology(FTLDU).To examine protein aggregation and cytotoxicity,we expressed human FUS protein in yeast.Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity,with the two ALS mutants showing increased cytotoxicity.Therefore,yeast cells expressing human FUS protein recapitulate key features of FUSpositive neurodegenerative diseases.Interestingly,a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates,suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity.The N-terminus of FUS,containing the QGSY and G rich regions,is sufficient for the formation of aggregates but not cytotoxicity.The C-terminal domain,which contains a cluster of mutations,did not show aggregation or cytotoxicity.Similar to TDP-43 when expressed in yeast,FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins.On the other hand,the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5%sarkosyl,unlike TDP-43 when expressed in yeast cells.Furthermore,TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.

Solution pH jump during antibody and Fc-fusion protein thaw leads to increased aggregation

Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization, There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted immunogenicity in patients, Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution, Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar effects were observed. The results provide a new tool to study the kinetics of therapeutic protein ag- gregation upon pH increase,

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.

Protein aggregation in association with delayed neuronal death in rat model of brain ischemia

To investigate the relationship between protein aggregation and delayed neuronal death,we adopted rat models of 20 min ischemia.Brain ischemia was produced using the 2-vessel occlusion(2VO)model in rats Light microscopy,transmission electronic microscopy and Western blot analysis were performed for morphological analysis of neurons,and protein detection.The results showed delayed neuronal death took place at 72 h after ischemia-reperfusion,protein aggregates formed at 4 h after reperfusion and reached the peak at 24 h after reper-fusion,and Western blot analysis was consistent with transmission electronic microscopy.We conclude that protein aggregation is one of the important factors leading to delayed neuronal death.

Systematic Analysis of Post-Translational Modifications for Increased Longevity of Biotherapeutic Proteins

Protein-based therapeutics (PPTs) are drugs used to treat a variety of different conditions in the human body by alleviating enzymatic deficiencies, augmenting other proteins and drugs, modulating signal pathways, and more. However, many PPTs struggle from a short half-life due to degradation caused by irreversible protein aggregation in the bloodstream. Currently, the most researched strategies for improving the efficiency and longevity of PPTs are post-translational modifications (PTMs). The goal of our research was to determine which type of PTM increases longevity the most for each of three commonly-used therapeutic proteins by comparing the docking scores (DS) and binding free energies (BFE) from protein aggregation and reception simulations. DS and BFE values were used to create a quantitative index that outputs a relative number from −1 to 1 to show reduced performance, no change, or increased performance. Results showed that methylation was the most beneficial for insulin (p < 0.1) and human growth hormone (p < 0.0001), and both phosphorylation and methylation were somewhat optimal for erythropoietin (p < 0.1 and p < 0.0001, respectively). Acetylation consistently provided the worst benefits with the most negative indices, while methylation had the most positive indices throughout. However, PTM efficacy varied between PPTs, supporting previous studies regarding how each PTM can confer different benefits based on the unique structures of recipient proteins.

Chiral LVFFARK enantioselectively inhibits amyloid-β protein fibrillogenesis

The modulation of protein aggregation is involved not only in biochemical engineering processes,but also in in vivo biological events such as Alzheimer's disease(AD)that features amyloid-βprotein(Aβ)deposits.Inspired by the different pharmacological efficacy of enantiomers,taking heptapeptide LVFFARK(LK7)as an example,herein the chiral influence of peptide inhibitors on Aβfibrillogenesis and cytotoxicity was investigated by extensive biophysical and biological analyses.It was intriguing to find that although both LLK7 and D-LK7 could inhibit Aβaggregation in a concentration-dependent manner,it was the D-enan-tiomer that exhibited chirality preference and selectivity for modulation of Aβself-assembly.As com-pared with L-LK7 at the same conditions,D-LK7 showed significantly enhanced potency on suppressing cross-βsheet formation,fibrillar Aβaggregates deposition,Aβconformational transition,and Aβ-triggered neurotoxicity on cultured cells.For instance,L.LK7 and D-LK7 rescued cells by increasing cell via-bility from 60%to 62%and 84%at 100μmolL^(-1),respectively.The chiral discrimination of L-LK7 and D-LK7 was further validated by the different elimination efficiency on amyloid accumulation in AD model nematodes.It is considered that the higher binding affinity of D-LK7 to Aβmonomers than that of L LK7 resulted in the stronger inhibition effect.This work provided new insights into understanding chiral-ity in the interaction with Aβand the consequent inhibitory effect,and would contribute to the design of anti-amyloid agents.

Insights into Late Embryogenesis Abundant (LEA) Proteins in Plants: From Structure to the Functions

Late Embryogenesis Abundant (LEA) proteins, a group of hydrophilic proteins, have been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. In this paper, we summarize and review research discoveries of the classification of the LEA protein groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. Moreover, we focus on high correlation between their accumulation and water deficit, reinforcing their functional relevance under abiotic stresses. We also discuss the biochemical properties of LEA proteins arising from their hydrophilic nature and by amino acid composition. Although significant similarities have not been found between the members of the different groups, a unifying and outstanding feature of most of them is their high hydrophilicity and high content of glycine. Therefore, we have highlighted the biotechnological applications of LEA genes, and the effects of over-expressing LEA genes from all LEA groups from different species of origin into different plant hosts. Apart from agronomical purposes, LEA proteins could be useful for other biotechnological applications in relation to their capacity to prevent aggregation of proteins.

One ring is sufficient to inhibit α-synuclein aggregation

Parkinson's disease,the second most prevalent neurodegenerative disorder worldwide,is characterized by a progressive loss of dopaminergic neurons in substantia nigra pars compacta,causing motor symptoms.This disorder's main hallmark is the formation of intraneuronal protein inclusions,named Lewy bodies and neurites.The major component of these arrangements is α-synuclein,an intrinsically disordered and soluble protein that,in pathological conditions,can form toxic and cell-to-cell transmissible amyloid structures.Preventing α-synuclein aggregation has attracted significant effort in the search for a disease-modifying therapy for Parkinson's disease.Small molecules like Synu Clean-D,epigallocatechin gallate,trodusquemine,or anle138 b exemplify this therapeutic potential.Here,we describe a subset of compounds containing a single aromatic ring,like dopamine,ZPDm,gallic acid,or entacapone,which act as molecular chaperones against α-synuclein aggregation.The simplicity of their structures contrasts with the complexity of the aggregation process,yet the block efficiently α-synuclein assembly into amyloid fibrils,in many cases,redirecting the reaction towards the formation of non-toxic off-pathway oligomers.Moreover,some of these compounds can disentangle mature α-synuclein amyloid fibrils.Their simple structures allow structure-activity relationship analysis to elucidate the role of different functional groups in the inhibition of α-synuclein aggregation and fibril dismantling,making them informative lead scaffolds for the rational development of efficient drugs.

RNA binding protein BOULE forms aggregates in mammalian testis

Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration.New evidence however increasingly suggests that non-pathological amyloids are formed in animals during normal development.Amyloid-like aggregate formation was originally thought to be a conserved feature of animal gametogenesis.This hypothesis was based on findings which suggest that regulated amyloid formations govern yeast meiosis by way of meiosis-specific RNA binding proteins.Additional support came from studies which demonstrate that DAZL,a mammalian gametogenesis-specific RNA binding protein,also forms SDS-resistant aggregates in vivo.Here,we report evidence of aggregated BOULE formations,another DAZ family protein,during sperm development.Data suggest that in mouse testis,BOULE forms SDS-resistant amyloid-like aggregates.BOULE aggregate formation correlates with dynamic developmental expression during spermatogenesis but disappeared in Boule knockout testis.We also mapped essential small region in vitro BOULE aggregations,immediately downstream DAZ repeats,and found that aggregations positively correlated with temperature.We also performed enhanced UV cross-linking immunoprecipitation on BOULE aggregates from mouse testes and found that aggregates bind with a large number of spermatogenesis-related mRNAs.These findings provide insight into the amyloidogenic properties of gametogenesis-specific RNA binding proteins as a conserved feature in mammalian reproduction.Further investigation is warranted to understand the functional significance of BOULE amyloid-like formation during mouse spermatogenesis.

Aggregation Patterns of Proteasome in Injured Neurons Induced by Transient Cerebral Ischemia

Proteasome activity reduction is an important pathological phenomenon, resulting in proteins aggregation and neuronal death in the injured neurons induced by transient ischemia. Our previous report showed that the trap of proteasome in the protein aggregates was a reason to lead to the reduction of proteasome activity. However, the patterns of proteasome entered into protein aggregates are not clear. In this study, we used a global ischemia model, Hematoxylin-Eosin staining, differential centrifuge, proteasome activity assay, sucrose gradient density centrifuge, and Western blot analysis to investigate this problem. Our results show that there are two aggregation patterns of proteasome after transient ischemia and reperfusion. One is that 26S proteasome is trapped by protein aggregates as a whole unit, and the other is that 19S or 20S is trapped in the protein aggregates, respectively, after 26S disassociates.

Natural polyphenols effects on protein aggregates in Alzheimer＇s and Parkinson＇s prion-like diseases

Alzheimer＇s and Parkinson＇s diseases are the most common neurodegenerative diseases. They are characterized by protein aggregates and so can be considered as prion-like disease. The major components of these deposits are amyloid peptide and tau for Alzheimer＇s disease, α-synuclein and synphilin-1 for Parkinson＇s disease. Drugs currently proposed to treat these pathologies do not prevent neurodegenerative processes and are mainly symptomatic therapies. Molecules inducing inhibition of aggregation or disaggregation of these proteins could have beneficial effects, especially if they have other beneficial effects for these diseases. Thus, several natural polyphenols, which have antioxidative, anti-inflammatory and neuroprotective properties, have been largely studied, for their effects on protein aggregates found in these diseases, notably in vitro. In this article, we propose to review the significant papers concerning the role of polyphenols on aggregation and disaggregation of amyloid peptide, tau, α-synuclein, synphilin-1, suggesting that these compounds could be useful in the treatments in Alzheimer＇s and Parkinson＇s diseases.

Consilience and unity in ocular anterior segment research

In his beautiful book,Consilience:The Unity of Knowledge,the eminent biologist Edward O Wilson,advocates the need for integration and reconciliation across the sciences.He defines consilience as“literally a‘jumping together’of knowledge with a linking of facts…to create a common groundwork of explanation”.It is the premise of this paper that as much as basic biomedical research is in need of data generation using the latest available techniques–unifying available knowledge is just as critical.This involves the necessity to resolve contradictory findings,reduce silos,and acknowledge complexity.We take the cornea and the lens as case studies of our premise.Specifically,in this perspective,we discuss the conflicting and fragmented information on protein aggregation,oxidative damage,and fibrosis.These are fields of study that are integrally tied to anterior segment research.Our goal is to highlight the vital need for Wilson’s consilience and unity of knowledge which in turn should lead to enhanced rigor and reproducibility,and most importantly,to greater understanding and not simply knowing.

Extracellular quality control in the epididymis

The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatinrelated epididymal spermatogenic （CRES） oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis. （Asian J Androl 2007 July; 9： 500-507）

One-pot in-situ synthesis of coralloid supported VO_(2)catalyst for intensified aerobic oxidative desulfurization

A coralloid 3D g-C_(3)N_(4)supported VO_(2)catalyst was successfully synthesized in-situ by one-pot method,avoiding the agglomeration of VO_(2)during the reaction.The morphological and compositional information of the supported catalyst were investigated detailedly.30%VO_(2)/3D g-C_(3)N_(4)revealed excellent catalytic activity in aerobic oxidative desulfurization,the oxidative of dibenzothiophene(DBT),4-methyldibenzothiophene(4-MDBT)and 4,6-dimethyldibenzothiophene(4,6-DMDBT)reached 98.6%,99%and 99.4%,respectively,under the same mild conditions.The recycling performance and the mechanism on the oxidative of DBT were studied as well.

α-Synuclein oligomers and fibrils:partners in crime in synucleinopathies

The misfolding and aggregation of a-synuclein is the general hallmark of a group of devastating neurodegenerative pathologies referred to as synucleinopathies,such as Parkinson’s disease,dementia with Lewy bodies,and multiple system atrophy.In such conditions,a range of different misfolded aggregates,including oligomers,protofibrils,and fibrils,are present both in neurons and glial cells.Growing expe rimental evidence supports the proposition that solu ble oligomeric assemblies,formed during the early phases of the aggregation process,are the major culprits of neuronal toxicity;at the same time,fibrillar confo rmers appear to be the most efficient at propagating among interconnected neurons,thus contributing to the spreading ofα-synuclein pathology.Moreover,α-synuclein fibrils have been recently repo rted to release soluble and highly toxic oligomeric species,responsible for an immediate dysfunction in the recipient neurons.In this review,we discuss the current knowledge about the plethora of mechanisms of cellular dysfunction caused byα-synuclein oligome rs and fibrils,both contributing to neurodegeneration in synucleinopathies.

Shiitake-derived lentinan preventsα-synuclein aggregation,disassembles amyloid aggregates,and protects dopaminergic neurons

Parkinson’s disease(PD)is the second largest degenerative disease of central nervous system around the world.α-Synuclein(α-syn)is the main component of Lewy body amyloid deposits in the brain of PD patients and associated with the pathogenesis of PD.α-Syn aggregates can induce neuron death and cause irreversible neurological damage.Therefore,the inhibition ofα-syn aggregation and removal of preformed amyloid fibers are important strategies for the prevention and treatment of PD.However,there is still a lack of potent inhibitors to interfere withα-syn aggregation.In this work,we demonstrate that lentinan(LNT),a natural polysaccharide extracted from the fruit body of Lentinula edodes,can potently preventα-syn aggregation,disassemble preformed amyloid aggregates,and suppressα-syn-mediated cell toxicity and reactive oxygen species generation.Interestingly,we also find that the molecular weight of the polysaccharide impacts the inhibitory effect of LNT.Further in vivo investigation demonstrates that LNT significantly inhibits the accumulation ofα-syn in nematodes C.elegans,relieves the symptoms of PD nematode motility disorders,and effectively protects dopaminergic neurons from C.elegans PD models.To the best of our knowledge,this is the first time to report that LNT can modulateα-syn aggregation in vitro and in vivo,which will provide new insights for designing and developing potential agents for PD treatment.

Tackling pesticide overusing through foliar deposition and retention:Frontiers and challenges

Pesticides are typically used excessively to ensure crop yields due to their low utilization extent,which can exert pressure on the environment and cause adverse effects on non-target species as well as pose a threat to human health.Hence,reducing the potential risks due to excessive pesticide use is an important goal of agriculture and environmental policies worldwide.The low pesticide utilization efficiency is attributed to poor droplet deposition on plant surfaces as well as weak interaction between pesticide and plant surfaces.In the past decades,considerable efforts have been put into solving these problems.However,a number of key challenges still remain to be addressed for the practical application.In this paper,we outline the development of a range of methods for improving the retention of pesticide on plant leaves.We also discuss and highlight several key issues that merit more attention in the future.

Peptide backbone-copper ring structure:A molecular insight into copper-induced amyloid toxicity

Copper ions can promote amyloid diseases that are associated with amyloid peptides, such as type 2 diabetes(T2D),Alzheimer's disease(AD), Parkinson's disease(PD), and amyotrophic lateral sclerosis(ALS). However, the underlying molecular mechanism remains obscure. Here we present that Cu^(2+)is able to specifically bind to the backbone of T2D related human islet amyloid polypeptide(hIAPP) by forming a ring structure, which causes the reduction of Cu^(2+)to Cu^(+) to produce reactive oxygen species(ROS) and the modulation of hIAPP aggregation. Nuclear magnetic resonance spectroscopy showed that Cu^(2+)bound to the backbone of a turn region, His18-Ser21, which is critical for hIAPP aggregation.Ab initio calculations and x-ray absorption fine structure analyses revealed that Cu^(2+)simultaneously bound with both the amide nitrogen and carbonyl oxygen on the peptide backbone, resulting in a ring structure, and causing the reduction of Cu^(2+)to Cu^(+) to form a hIAPP-Cu^(+) complex. 2′,7′-dichlorodihydrofluorescin diacetate fluorescence measurements further indicated that this complex led to enhanced ROS levels in rat insulinoma cells. Additionally, thioflavin T fluorescence and atomic force microscopy measurements denoted that the backbone-Cu ring structure largely modulated hIAPP aggregation,including the inhibition of hIAPP fibrillation and the promotion of peptide oligomerization. These findings shed new light on the molecular mechanism of Cu^(2+)-induced amyloid toxicity involving both the enhancement of ROS and the modulation of hIAPP aggregation.