Characterization of an antibody scFv that recognizes fibrillar insulin and beta-amyloid using atomic force microscopy

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.     

Mechanistic studies of the process of amyloid fibrils formation by the use of peptide fragments and analogues: implications for the design of fibrillization inhibitors

The process of amyloid fibrils formation is a common mechanism of a large number of unrelated infectious, genetic and spontaneous diseases. A partial list includes the bovine spongiform encephalopathy (BSE), Alzheimer's diseases, Type II diabetes, Creutzfeldt-Jakob disease, and various unrelated amyloidosis diseases. In spite of its significant clinical importance, the mechanism of fibrillization is not fully understood. This review discusses the recent advancements in the mechanistic studies of amyloid formation by the use peptide fragments and analogues of amyloid-forming proteins and polypeptides. The use of short peptide shed much light of the mechanism of amyloid fibrillization. Recent studies clearly prove that very short peptide fragments (as short as pentapeptides) can form well-ordered amyloidal structures. Therefore, the molecular recognition and self-assembly process that lead to the formation of order structures is being mediated by small structural elements. Analysis of short amyloid-related fragment by the use of an alanine-scan and sequence analysis of a variety of unrelated peptide and protein fragments suggest that aromatic interaction may play a central role in the process of amyloid formation. Inhibitors that are based on the short aromatic elements already demonstrated clear potency in arresting the process of amyloid fibrils formation. Taken together, the recent advancement in the mechanistic understanding of the process of amyloid fibrils formation has a major importance in the development of inhibitors of fibrillization that may serve as future therapeutic means to treat amyloid diseases.     

Turmeric effect on subcutaneous insulin-induced amyloid mass: an in vivo study

Protein-derived amyloid structures are associated with a wide variety of pathologies, including neurodegenerative diseases and local amyloidoses. Reports exist on the ability of insulin to form local amyloidoses under specific conditions. In vitro-generated fibrils of insulin have been previously shown to produce amyloid-containing masses upon repetitive subcutaneous injection in mouse. The present study aimed at investigating the effect of insulin fibrils injection in rats, as well as the potential of turmeric in attenuating this process. It was found that subcutaneous amyloid-containing masses could form in rats at a faster rate compared with mice. Upon addition of turmeric to the fibrils, previous to injection, formed masses had a significantly reduced size, as well as less ordered cellular structure. In conclusion, the results of this study show the potential of turmeric in attenuation of local amyloidosis. Furthermore, we suggest that this model could be of use in screening antiamyloid compounds.     

Inhibition of aggregate formation as therapeutic target in protein misfolding diseases: effect of tetracycline and trehalose

Importance of the field: The identification of molecules that inhibit protein deposition or reverse fibril formation could open new strategies for therapeutic intervention in misfolding diseases. Numerous compounds have been shown to inhibit amyloid fibril formation in vitro. Among these compounds, tetracycline and the disaccharide trehalose have been reported to inhibit or reverse amyloid aggregation but their efficiency as potential drugs is controversial.

Areas covered in this review: The results obtained using tetracycline and trehalose, reported in the last 15 years, are described and discussed.

What the reader will gain: The conclusions have important implications for the development of therapeutic agents for protein deposition diseases. If fibrillar proteins contribute to cell degeneration, then the disassembly of fibrils may reverse or slow down disease progression; however, if the action of therapeutic agents produces intermediates of fibrillation and/or products of fibril disaggregation, then their accumulation could be harmful.

Take home message: Care should be taken to ensure that strategies aimed at inhibiting fibril formation do not cause a corresponding increase in the concentration of toxic oligomeric species.     

Aggregation of the neuroblastoma-associated mutant (S120G) of the human nucleoside diphosphate kinase-A/NM23-H1 into amyloid fibrils

The human nucleoside diphosphate (NDP) kinase A, product of the NME1 gene also named NM23-H1, is known as a metastasis suppressor protein. A naturally occurring variant, S120G, identified in neuroblastomas, possesses native three-dimensional structure and enzymatic activity but displays reduced conformational stability and a folding defect with the accumulation of a "molten globule" folding intermediate during refolding in vitro. As such intermediate has been postulated to be involved in amyloid formation, NDP kinase A may serve as a model protein for studying the relationship between folding intermediates and amyloid fibrils. The NDP kinase A S120G was heated in phosphate buffer (pH?7.0). The protein precipitated as amyloid fibrils, as demonstrated by electron microscopy, Congo red, and thioflavin T binding and FTIR spectroscopy. The NDP kinase A S120G, at neutral pH and at moderate temperature experiences a transition towards amyloid fibrils. The aggregation process was faster if seeded by preformed fibrils. The fibrils presented a large proteinase K-resistant core not including residue Gly 120, as shown by mass spectrometry. This suggests that the aggregation process is triggered by the reduced stability of the S120G variant and not by a specific increase in the kinase domain intrinsic aggregation propensity at the place of mutation. This constitutes one of the few reports on a protein involved in cancer biology able to aggregate into amyloid structures under mild conditions.     

Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.     

芦丁对β淀粉样肽纤维化及细胞毒性的抑制作用

目的探索芦丁对Aβ25-35肽段淀粉样纤维化及纤维细胞毒性的抑制作用。方法在pH值为7.4、温度37℃孵育Aβ25-35肽,采用硫黄素(thioflavin T,ThT)荧光和透射电子显微镜检测多肽的淀粉样纤维化;以淀粉样纤维处理PC12细胞建立的细胞损伤模型,MTT法检测细胞存活率,以评估芦丁对β淀粉样纤维细胞毒性的抑制作用。结果 Aβ25-35肽段在pH值为7.4、温度37℃条件下,经孵育60h左右形成淀粉样纤维;芦丁抑制Aβ淀粉样纤维的形成,破坏纤维结构,并降低纤维诱导的细胞损害。结论芦丁能够抑制Aβ25-35的淀粉样纤维化和破坏成熟纤维结构,并降低Aβ纤维的细胞毒性。     

Amyloidogenesis in Alzheimer's disease: some possible therapeutic opportunities.

Cerebral deposition of fibrils formed from the beta/A4 amyloid protein is an invariable feature of Alzheimer's disease. Evidence suggests that generation of such fibrils may be involved in the etiology of this disease, since mutations in the coding region of the beta/A4 amyloid precursor protein (APP) gene segregate with familial cerebral amyloidoses, including familial Alzheimer's disease. Transgenic models of cerebral amyloidosis have been produced, and some progress has been made in elucidating the cell biology of amyloidogenesis. For example, agents that alter protein phosphorylation are potent modulators of the expression and proteolytic processing of APP. Sam Gandy and Paul Greengard review these recent studies, and discuss those that may provide rational therapeutic opportunities.     

聚乙二醇对溶菌酶淀粉样纤维形成的作用

目的探索在聚乙二醇(PEG)存在的条件下,溶菌酶淀粉样纤维化及由此生成的聚集体对细胞的毒性作用。方法溶菌酶溶液中分别加入不同相对分子质量的PEG,在pH值为2.0、55℃的条件下孵育。采用硫黄素荧光监测溶菌酶淀粉样纤维化,透射电子显微镜观察聚集体形态,以诱导人红细胞聚集和溶血为指标评估溶菌酶聚集体对细胞膜的损害作用。结果在实验条件下,溶菌酶可形成淀粉样纤维,所有PEG均能够抑制溶菌酶的淀粉样纤维化,改变溶菌酶聚集体的形态,降低聚集体的表面疏水性和聚集体对细胞的损害作用。结论 PEG能够抑制溶菌酶的淀粉样纤维化,并使溶菌酶聚集体的细胞毒性减弱。     

Pharmaceutical strategies against amyloidosis: old and new drugs in targeting a "protein misfolding disease"

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, protein's post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.     

Synthesis and amyloid binding properties of rhenium complexes: preliminary progress toward a reagent for SPECT imaging of Alzheimer's disease brain.

The definitive diagnosis of Alzheimer's disease (AD) requires the detection of amyloid plaques in postmortem brain. Although the amount of fibrillar amyloid roughly correlates with the severity of symptoms at the time of death, the temporal relationship between amyloid deposition, neuronal loss, and cognitive decline is unclear. To elucidate this relationship, a noninvasive, practical method for the quantitation of brain amyloid deposition is required. We describe herein the initial stages of a strategy to accomplish this goal by single photon computed tomographic imaging. The amyloid-binding dye Congo Red was modified to allow its conjugation to the monoamine-monoamide bis(thiol) ligand. This ligand complexes technetium(V) in its neutral oxo form. A biphenyl-containing building block was conjugated to the protected ligand, and the product was coupled to the relevant aromatic compounds. Rhenium oxo complexes, which are isosteric, but nonradioactive, analogues of the potential imaging agent technetium oxo complexes, were synthesized. These complexes bound to Abeta amyloid fibrils produced in vitro and stained amyloid plaques and vascular amyloid in AD brain sections.     

Amyloid associated proteins in Alzheimer's and prion disease

Clustering of activated microglia in Abeta deposits is related to accumulation of amyloid associated factors and precedes the neurodegenerative changes in AD. Microglia-derived pro-inflammatory cytokines are suggested to be the driving force in AD pathology. Inflammation-related proteins, including complement factors, acute-phase proteins, pro-inflammatory cytokines, that normally are locally produced at low levels, are increasingly synthesized in Alzheimer's disease (AD) brain. Similar to AD, in prion diseases (Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker disease and experimentally scrapie infected mouse brain) amyloid associated factors and activated glial cells accumulate in amyloid deposits of conformational changed prion protein (PrPres). Biological properties of Abeta and prion (PrP) peptides, including their potential to activate microglia, relate to Abeta and PrP peptide fibrillogenic abilities that are influenced by certain amyloid associated factors. However, since small oligomers of amyloid forming peptides are more toxic to neurons than large fibrils, certain amyloid associated factors that enhance fibril formation, may sequester the potentially harmful Abeta and PrP peptides from the neuronal microenvironment. In this review the positive and negative actions of amyloid associated factors on amyloid peptide fibril formation and on the fibrillation state related activation of microglia will be discussed. Insight in these mechanisms will enable the design of specific therapies to prevent neurodegenerative diseases in which amyloid accumulation and glial activation are prominent early features.     

β-Amyloid peptide as a target for treatment of Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative disorder characterised by a series of biochemical and histological changes although the net of relations and its initial cause is far from being fully understood. The amyloid hypothesis points out the pathological processing of a physiologically normal protein, the amyloid precursor protein, to neurotoxic forms of amyloid β-peptide as the origin of the cascade of biochemical changes that lead to Alzheimer’s disease. Normal APP processing involves three proteases, α-, β- and γ-secretase, to yield physiological amyloid fragments. Familial Alzheimer’s disease patients exhibit an increased activity of β- and γ-secretases, resulting in higher than average levels of small amyloid fragments, of 40 or 42 amino acids (Aβ₄₀ and Aβ₄₂, respectively). These newly formed Aβ₄₀ and Aβ₄₂ may suffer a conformational change followed by aggregation into fibrils and finally deposition as senile plaques in a complex process named fibrillogenesis, which is associated with neurotoxicity. Modulation of this multistep process is a reasonably hopeful approach for the treatment of Alzheimer’s disease. In a general sense, this approach can be divided in three groups: first, modulating the production of Aβ promoting the non-amyloidogenic route; second, inhibiting fibrillogenesis and third, by immunisation techniques, enhancing the formation of anti-Aβ antibodies in order to mark fibrils and plaques as targets for microglial cells.     

Influence of EGCG on α‐synuclein (αS) aggregation and identification of their possible binding mode: A computational study using molecular dynamics simulation

The accumulation of intrinsically disordered α‐synuclein (αS) protein that can form β‐sheet‐rich fibrils is linked to Parkinson's disease. (−)‐Epigallocatechin‐3‐gallate (EGCG) is the most abundant active component in green tea and can inhibit the fibrillation of αS. The elucidation of this molecular mechanism will be helpful to understand the inhibition mechanism of EGCG to the fibrillation of αS and also to find more potential small molecules that can inhibit the aggregation of αS. In this work, to study the influence of EGCG on the structure of β‐sheet‐rich fibrils of αS and identification of their possible binding mode, molecular dynamics simulations of pentamer and decamer aggregates of αS in complex with EGCG were performed. The obtained results indicate that EGCG can remodel the αS fibrils and break the initial ordered pattern by reducing the β‐sheet content. EGCG can also break the Greek conformation of αS by the disappeared H‐bond in the secondary structure of turn. The results from our study can not only reveal the specific interaction between EGCG and β‐sheet‐rich fibrils of αS, but also provide the useful guidance for the discovery of other potential inhibitors.     

The role of heparan sulfate in the generation of Abeta

Alzheimer's disease is a fatal neurodegenerative disorder for which there are currently few treatments and no cure. Heparan sulfate, a heterogeneously sulfated glycosaminoglycan, has been identified as the first naturally occurring inhibitor of beta secretase, the rate-limiting step in the formation of Abeta, the peptide core of the amyloid plaques that cause Alzheimer's disease. Though heparan sulfate has frequently been implicated in the formation of fibrils, only fairly recently has its role as an inhibitor of beta secretase been recognized. This inhibitory activity is dependent on the structure and size of the heparan sulfate chain, with emphasis placed on the position of the sulfates. Heparan sulfate directly binds to beta secretase and causes a closed configuration of the catalytic site. Regulation of amyloid precursor protein (APP) beta secretase cleavage could occur at a number of cellular locations, including the Golgi complex, endosomal system and cell surface. Heparan sulfate also binds to APP and may sequester it away from beta secretase. These findings have led to the examination of heparan sulfate analogues, such as beta-secretase inhibitors, as a potential therapeutic approach to treat Alzheimer's disease.     

Insulin Formulation Characterization—the Thioflavin T Assays

The insulin molecule was discovered in 1921. Shortly thereafter, its propensity towards amyloid fibril formation, fibrillation, was observed and described in the literature as a “precipitate.” In the past decades, the increased incidence of type 2 diabetes has reached global epidemic proportions. This has emphasized the demands for both insulin production and the development of modern insulin products for unmet medical needs. Bringing such new insulin drug products to the market for the benefit of patients requires that many CMC-related processes are understood, described, and controlled. One potential undesired process is insulin fibril formation. The compound thioflavin T (ThT) is known as a fluorescent probe for amyloid fibrils. As such, ThT is utilized in a versatile research assay in microtiter plate format, the ThT assay. This review will describe an experimental set-up using not only a ThT microtiter plate assay but also two orthogonal methods. The use of the ThT assay in research and characterization of insulin analogues, as well as formulations of insulin, is described by cases drawn from the scientific literature and patents. The ThT assay is compared to other physical stability tests and in conclusion the advantages and limitations of the assay are compared.     

Syntheses and structure-activity relationships of seven manganese-salen derivatives as anti-amyloidogenic and fibril-destabilizing agents against hen egg-white lysozyme aggregation

Accumulation of intra- and/or extracellular misfolded proteins as amyloid fibrils is a key hallmark in more than 20 amyloid-related diseases. In that respect, blocking or reversing amyloid aggregation via the use of small compounds is considered as two useful approaches in hampering the development of these diseases. In this research, we have studied the ability of different manganese-salen derivatives to inhibit amyloid self-assembly as well as to dissolve amyloid aggregates of hen egg-white lysozyme, as an in vitro model system, with the aim of investigating their structure-activity relationships. By coupling several techniques such as thioflavin T and anilinonaphthalene-8-sulfonic acid fluorescence, congo red absorbance, far-UV circular dichroism, and transmission electron microscopy, we demonstrated that all compounds possessed anti-amyloidogenic activities and were capable of dispersing the fibrillar aggregates. In addition, MTT assay of the treated SK-N-MC cells with the preformed fibrils formed in the presence of compounds at a drug-to-protein molar ratio of 5:1, indicated a significant increase in the viability of cells, compared to the fibrils formed in the absence of each of the compounds. Our spectroscopy, electron microscopy, and cellular studies indicated that EUK-15, with a methoxy group at the para position (group R(5)), had higher activity to either inhibit or disrupt the β-sheet structures relative to other compounds. On the basis of these results, it can be concluded that in addition to aromatic rings of each of the derivatives, the type and position of the side group(s) contribute to lower lysozyme fibril accumulation.     

槲皮素对淀粉样纤维细胞毒性的抑制作用

目的采用人红细胞为实验模型,探索槲皮素对溶菌酶淀粉样纤维细胞毒性的抑制作用。方法制备溶菌酶淀粉样纤维,在溶菌酶纤维溶液中加入槲皮素,用原子力显微镜观察槲皮素对淀粉样纤维的分解作用;在红细胞悬液中加入溶菌酶淀粉样纤维和槲皮素,用扫描电镜观察细胞形态;SDS凝胶电泳分离细胞膜蛋白,检测在槲皮素存在的条件下,溶菌酶纤维诱导膜蛋白聚集的作用。结果槲皮素能够破坏淀粉样纤维结构,使纤维解聚,从而使溶菌酶淀粉样纤维的细胞损害作用降低,包括抑制溶菌酶纤维诱导的细胞聚集和细胞膜蛋白交联。结论槲皮素能够破坏成熟的溶菌酶淀粉样纤维结构,抑制淀粉样纤维对细胞膜的损害作用。槲皮素的这种作用与其分子的疏水性和抗氧化作用有关。     

Amyloid disease prevention by transthyretin native state complexation with carborane derivatives lacking cyclooxygenase inhibition

Misfolding and subsequent aggregation of any of a number of proteins leads to the accumulation of amyloid fibrils, which have been associated with a variety of diseases. One such amyloidogenic protein is transthyretin (TTR), a 55-kDa homotetrameric protein found in the blood plasma and cerebrospinal fluid where it binds and transports thyroxine. In humans, the T119M-TTR variant has been shown to be protective against familial amyloid polyneuropathy, a TTR amyloid disease, through kinetic stabilization of the unliganded tetrameric structure. Studies have indicated that a diverse range of small molecules may also bind TTR in the thyroxine-binding pocket and subsequently kinetically stabilize the protein's native conformation in vitro, preventing the misfolding that has been implicated in the progression of several diseases. However, cyclooxygenase inhibition is a common unwanted side effect among such small-molecule kinetic stabilizers. The recent development of transthyretin stabilizers not subject to cyclooxygenase inhibition may prove attractive for the long-term treatment of TTR misfolding diseases in humans. Such compounds are attained by incorporating aromatic carborane icosahedra at strategic points in their structures.     

A novel beta-sheet breaker,RS-0406, reverses amyloid beta-induced cytotoxicity and impairment of long-term potentiation in vitro

Fibril formation of amyloid beta peptide (Abeta) is considered to be responsible for the pathology of Alzheimer's disease (AD). The Abeta fibril is formed by a protein misfolding process in which intermolecular beta-sheet interactions become stabilized abnormally. Thus, to develop potential anti-AD drugs, we screened an in-house library to find compounds which have a profile as a beta-sheet breaker. We searched for a beta-sheet breaker profile in an in-house library of approximately 113,000 compounds. From among the screening hits, we focused on N,N'-bis(3-hydroxyphenyl)pyridazine-3,6-diamine (named RS-0406), which had been newly synthesized in our laboratory. This compound (10-100 microg ml(-1)) was found to be capable of significantly inhibiting 25 microM Abeta(1-42) fibrillogenesis and, furthermore, disassembling preformed Abeta(1-42) fibrils in vitro. 3 We then investigated the effect of RS-0406 on 111 nM Abeta(1-42)-induced cytotoxicity in primary hippocampal neurons, and found that 0.3-3 microg ml(-1) RS-0406 ameliorates the cytotoxicity. Moreover, 3 microg ml(-1) RS-0406 reversed 1 micro M Abeta(1-42)-induced impairment of long-term potentiation in hippocampal slices. 4 In this study, we have succeeded in identifying RS-0406 which has potential to inhibit Abeta(1-42) fibrillogenesis, and to protect neurons against Abeta(1-42)-induced biological toxicity in vitro. These results suggest that RS-0406 or one of the derivatives could become a therapeutic agent for AD patients.