Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (～ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.     

Characterization and immobilization of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti

A novel oligomeric SGNH hydrolase (Est24) from Sinorhizobium meliloti was identified, actively expressed in Escherichia coli, characterized, and immobilized for industrial application. Sequence analysis of Est24 revealed a putative catalytic triad (Ser¹³-Asp¹⁶³-His¹⁶⁹), with moderate homology to other SGNH hydrolases. Est24 was more active toward short-chain esters, such as p-nitrophenyl acetate, butyrate, and valerate, while the S13A mutant completely lost its activity. Moreover, the activity of Est24 toward α- and β-naphthyl acetate, and enantioselectivity on (R)- and (S)-methyl-3-hydroxy-2-methylpropionate were tested. Est24 exhibited optimum activity at mesophilic temperature ranges (45–55 °C), and slightly alkaline pH (8.0). Structural and mutagenesis studies revealed critical residues involved in the formation of a catalytic triad and substrate-binding pocket. Cross-linked enzyme aggregates (CLEAs) of Est24 with and without amyloid fibrils were prepared, and amyloid fibril-linked Est24 with amyloid fibrils retained 83 % of its initial activity after 1 h of incubation at 60 °C. The high thermal stability of immobilized Est24 highlights its potential in the pharmaceutical and chemical industries.     

Amyloid fibrils as a nanoscaffold for enzyme immobilization

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross‐linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD‐functionalized amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Inhibition of chaperonin GroEL by a monomer of ovine prion protein and its oligomeric forms

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.     

Dehydration stability of amyloid fibrils studied by AFM

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Aβ(1–42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Aβ(1–42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.     

Direct visualisation of the beta-sheet structure of synthetic Alzheimer's amyloid

Amyloid fibrils are a major pathological feature of Alzheimer's disease as well as other amyloidoses including the prion diseases. They are an unusual phenomenon, being made up of different, normally soluble proteins which undergo a profound conformational change and assemble to form very stable, insoluble fibrils which accumulate in the extracellular spaces. In Alzheimer's disease the amyloid fibrils are composed of the A beta protein. Knowledge of the structure of amyloid is essential for understanding the abnormal assembly and deposition of these fibrils and could lead to the rational design of therapeutic agents for their prevention or disaggregation. Here we reveal the core structure of an Alzheimer's amyloid fibril by direct visualisation using cryo-electron microscopy. Synthetic amyloid fibrils composed of A beta residues 11 to 25 and 1 to 42 were examined. The A beta (11-25) fibrils are clearly composed of beta-sheet structure that is observable as striations across the fibres. The beta-strands run perpendicular to the fibre axis and the projections show that the fibres are composed of beta-sheets with the strands in direct register. This observation has implications not only for the further understanding of amyloid, but also for the development of cryo-electron microscopy for direct visualisation of secondary structure.     

Ganglioside-induced amyloid formation by human islet amyloid polypeptide in lipid rafts

Human islet amyloid polypeptide (hIAPP) is the primary component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus. However, it is unknown how amyloid fibrils are formed in vivo. In this study, we demonstrate that gangliosides play an essential role in the formation of amyloid deposits by hIAPP on plasma membranes. Amyloid fibrils accumulated in ganglioside- and cholesterol-rich microscopic domains (‘lipid rafts’). The depletion of gangliosides or cholesterol significantly reduced the amount of amyloid deposited. These results clearly showed that the formation of amyloid fibrils was mediated by gangliosides in lipid rafts.     

Deposition of amyloid in the liver of hamsters: an enzyme-histochemical and electron-microscopical study.

In induced amyloidosis the amyloid was first deposited in the portal areas, then in the centre of the lobule and disseminated through it in the spaces of Disse. No intracellular amyloid was found in the predeposit phase. In the hepatic lobule both mononuclear phagocytes and hepatocytes showed a topographic relationship to the first deposits of amyloid. Some macrophages showed invaginations or vacuoles containing amyloid fibrils. Between the microvilli of hepatocytes, parallel amyloid fibrils occurred. Between the amyloid fibrils were 30-50 nm membrane-bounded spherical particles which may have been lipoprotein aggregates. There was no large-scale phagocytosis of amyloid during the induction period or after survival without casein treatment up to 18 weeks. Lysosomal enzyme activity was seen in the deposits of extracellular amyloid.     

A study on the interaction of the amyloid fibrils of α-synuclein and hen egg white lysozyme with biological membranes

Alpha-synuclein (α-syn) aggregation and mitochondrial dysfunction are considered as two of the main factors associated with Parkinson's disease (PD). In the present investigation, the effectiveness of the amyloid fibrils obtained from α-syn with those of hen egg white lysozyme (HEWL), as disease-related and-unrelated proteins, to damage rat brain and rat liver mitochondria have been investigated. This was extended by looking at SH-SY5Y human neuroblastoma cells and erythrocytes, thereby investigating the significance of structural characteristics of amyloid fibrils related to their interactions with biomembranes obtained from various sources. Results presented clearly demonstrate substantial differences in the response of tested biomembranes to toxicity induced by α-syn/HEWL amyloid fibrils, highlighting a structure-function relationship. We found that fibrillar aggregates of α-syn, but not HEWL, caused a significant increase in mitochondrial ROS, loss of membrane potential, and mitochondrial swelling, in a dose-dependent manner. Toxicity was found to be more pronounced in brain mitochondria, as compared to liver mitochondria. For SH-SY5Y cells and erythrocytes, however, both α-syn and HEWL amyloid fibrils showed the capacity to induce toxicity. Taken together, these results may suggest selective toxicity of α-syn amyloid fibrils to mitochondria mediated likely by their direct interaction with the outer mitochondrial membrane, indicating a correlation between specific structural characteristics of α-syn fibrils and an organelle strongly implicated in PD pathology.     

Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.     

Induction of AApoAII amyloidosis by various heterogeneous amyloid fibrils

Preformed amyloid fibrils accelerate conformational changes of amyloid precursor proteins and result in rapid extension of amyloid fibrils in vitro. We injected various kinds of amyloid fibrils into mice with amyloidogenic apoAII gene (Apoa2(C)). The most severe amyloid depositions were detected in the tissues of mice injected with mouse AApoAII(C) amyloid fibrils. Mild amyloid depositions were also detected in the tissues of mice that were injected with other types of fibrils, including synthetic peptides and recombinant proteins. However, no amyloid depositions were found in mice that were injected with non-amyloid fibril proteins. These results demonstrated that a common structure of amyloid fibrils could serve as a seed for amyloid fibril formation in vivo.     

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the β-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter- and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.     

UV-light exposed prion protein fails to form amyloid fibrils

Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Protein particulates: another generic form of protein aggregation?

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer's disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for beta-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.     

Insulin-degrading enzyme: embarking on amyloid destruction.

Several human disorders are caused by or associated with the deposition of protein aggregates known as amyloid fibrils. Despite the lack of sequence homology among amyloidogenic proteins, all amyloid fibrils share a common morphology, are insoluble under physiological conditions and are resistant to proteolytic degradation. Because amyloidogenic proteins are being produced continuously, eukaryotic organisms must have developed a form of proteolytic machinery capable of controlling these aggregation-prone species before their fibrillization. This article suggests that an intracellular metalloprotease called insulin-degrading enzyme (IDE) is responsible for the elimination of proteins with amyloidogenic potential and proposes a mechanism for the selectivity of the enzyme. In this respect, IDE can also be referred to as ADE: amyloid-degrading enzyme.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.