Rita P.-Y. Chen

Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.

Amyloid-Degrading Ability of Nattokinase from Bacillus subtilis Natto

More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimers disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.

Curcumin's pre-incubation temperature affects its inhibitory potency toward amyloid fibrillation and fibril-induced cytotoxicity of lysozyme.

BACKGROUND More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumins thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL). METHODS ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability. RESULTS AND CONCLUSION The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumins thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumins thermostability was associated with its inhibitory potency against HEWL fibrillation. GENERAL SIGNIFICANCE We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Modulation of RGD-functionalized polyelectrolyte multilayer membranes for promoting osteoblast function.

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) membranes has recently been applied successfully to a number of biomedical applications. This simple and versatile technique provides a broad surface modification platform, for example, for the display of biomolecules such as cell-adhesion peptides. In this work, we investigated the effects of PEM coatings on RGD-immobilization and osteoblast cell culture. RGD-containing peptides were conjugated to the amino groups of poly(allylamine hydrochloride) (PAH), and then adsorbed on top of 10-layer PAH/poly(acrylic acid) (PAA) multilayer membranes that were assembled at either pH 2.0 or pH 6.5. MG63 osteoblast-like cells were then seeded and cultured on the RGD-conjugated surfaces. We found that the cells adhered to and grew better on the RGD-conjugated PEM membranes. Furthermore, the cells grew better on the RGD-conjugated PEM coatings assembled at pH 6.5 than those assembled at pH 2.0. On the other hand, MG63 cells exhibited better differentiated phenotype on the pH 2.0 coatings compared to the pH 6.5 coatings with respect to alkaline phosphatase activity and calcium deposition, while cells did not express osteoblast phenotype on the PAH surfaces. These results clearly show that the base PEM membranes play an important role in RGD-immobilization and osteoblast functions.

Anterior development in the parthenogenetic and viviparous form of the pea aphid, Acyrthosiphon pisum: hunchback and orthodenticle expression

In the dipteran Drosophila, the genes bicoid and hunchback work synergistically to pattern the anterior blastoderm during embryogenesis. bicoid, however, appears to be an innovation of the higher Diptera. Hence, in some non‐dipteran insects, anterior specification instead relies on a synergistic interaction between maternally transcribed hunchback and orthodenticle. Here we describe how orthologues of hunchback and orthodenticle are expressed during oogenesis and embryogenesis in the parthenogenetic and viviparous form of the pea aphid, Acyrthosiphon pisum. A. pisum hunchback (Aphb) mRNA is localized to the anterior pole in developing oocytes and early embryos prior to blastoderm formation – a pattern strongly reminiscent of bicoid localization in Drosophila. A. pisum orthodenticle (Apotd), on the other hand, is not expressed prior to gastrulation, suggesting that it is the asymmetric localization of Aphb, rather than synergy between Aphb and Apotd, that regulates anterior specification in asexual pea aphids.

Developmental expression of Apnanos during oogenesis and embryogenesis in the parthenogenetic pea aphid Acyrthosiphon pisum

Among genes that are preferentially expressed in germ cells, nanos and vasa are the two most conserved germline markers in animals. Both genes are usually expressed in germ cells in the adult gonads, and often also during embryogenesis. Both nanos-first or vasa-first expression patterns have been observed in embryos, implying that the molecular networks governing germline development vary among species. Previously we identified Apvasa, a vasa homologue expressed in germ cells throughout all developmental stages in the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum. In asexual A. pisum, oogenesis is followed by embryogenesis, and both occur within the ovarioles. In order to understand the temporal and spatial distribution of nanos versus vasa during oogenesis and embryogenesis, we isolated a nanos homologue, Apnanos, and studied its expression. In adults, Apnanos is preferentially expressed in the ovaries. In early embryos, Apnanos transcripts are localized to the cytoplasm of cellularizing germ cells, and soon thereafter are restricted to the newly segregated germ cells in the posterior region of the cellularized blastoderm. These results strongly suggest that the Apnanos gene is a germline marker and is involved in germline specification in asexual A. pisum. However, during the middle stages of development, when germline migration occurs, Apnanos is not expressed in the migrating germ cells expressing Apvasa, suggesting that Apnanos is not directly associated with germline migration.

A highly sensitive peptide substrate for detecting two Aβ-degrading enzymes: Neprilysin and insulin-degrading enzyme

Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimers disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected.

A proposed role for Leishmania major carboxypeptidase in peptide catabolism

Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids--the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9-12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes--the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed.

Thioflavin T and Its Photoirradiative Derivatives: Exploring Their Spectroscopic Properties in the Absence and Presence of Amyloid Fibrils

In this work, we found that, during storage or after UV irradiation, ThT is demethylated or oxidized, forming three derivatives. These three derivatives were purified by high performance liquid chromatography and characterized by mass and nuclear magnetic resonance spectroscopy and the spectroscopic properties of pure ThT and the derivatives carefully compared. Our results show that the emission peak at 450 nm results from oxidized ThT and not from the monomeric form of ThT, as previously proposed. The partial conversion of ThT into oxidized and demethylated derivatives has an effect on amyloid detection using ThT assay. Irradiated ThT has the same lag time as pure ThT in the amyloidogenesis of insulin, but the intensity of the emitted fluorescence is significantly decreased.