The role of protein hydrophobicity in conformation change and self-assembly into large amyloid fibers.

It has been found that a short hydrophobic "template" peptide and a larger α-helical "adder" protein cooperatively self-assemble into micrometer sized amyloid fibers. Here, a common template of trypsin hydrolyzed gliadin is combined with six adder proteins (α-casein, α-lactalbumin, amylase, hemoglobin, insulin, and myoglobin) to determine what properties of the adder protein drive amyloid self-assembly. Utilizing Fourier Transform-Infrared (FT-IR) spectroscopy, the Amide I absorbance reveals that the observed decrease in α-helix with time is approximately equal to the increase in high strand density ß-sheet, which is indicative of amyloid formation. The results show that the hydrophobic moment is a good predictor of conformation change but the fraction of aliphatic amino acids within the α-helices is a better predictor. Upon drying, the protein mixtures form large amyloid fibers. The fiber twist is dependent on the aliphatic index and molecular weight of the adder protein. Here we demonstrate that it is possible to predict the propensity of an adder protein to unfold into an amyloid structure and to predict the fiber morphology, both from adder protein molecular features, which can be applied to the pragmatic engineering of large amyloid fibers.

Peptide mixtures can self-assemble into large amyloid fibers of varying size and morphology.

Peptide mixtures spontaneously formed micrometer-sized fibers and ribbons from aqueous solution. Hydrolyzed gliadin produced short, slightly elliptical fibers while hydrolyzed wheat gluten, a mixture of gliadin and glutenin, formed round fibers of similar size. Mixing hydrolyzed gliadin with increasing molar amounts of myoglobin or amylase resulted in longer, wider fibers that transitioned from round to rectangular cross section. Fiber size, morphology, and modulus were controlled by peptide mixture composition. Fourier transform infrared (FT-IR) spectroscopy results showed that peptides experienced α to ß transitions forming an elementary cross-ß peptide secondary structure, indicative of amyloids. Large fiber formation was observed to be dependent on hydrophobic packing between constituent peptides. A model was developed to show how the fiber morphology was influenced by the peptides in the mixture.

Phoenix: A Portable, Battery-Powered, and Environmentally Controlled Platform for Long-Distance Transportation of Live-Cell Cultures.

Despite the advent of advanced therapy medicinal products (ATMPs) in regenerative medicine, gene therapy, cell therapies, tissue engineering, and immunotherapy, the availability of treatment is limited to patients close to state-of-the-art facilities. The SCORPIO-V Division of HNu Photonics has developed the Phoenix-Live Cell TransportTM, a battery-operated mobile incubator designed to facilitate long-distance transportation of living cell cultures from GMP facilities to remote areas for increased patient accessibility to ATMPs. This work demonstrates that PhoenixTM (patent pending) is a superior mechanism for transporting living cells compared to the standard method of shipping frozen cells on dry ice (-80°C) or in liquid nitrogen (-150°C), which are destructive to the biology as well as a time consuming process. Thus, Phoenix will address a significant market need within the burgeoning ATMP industry. SH-SY5Y neuroblastoma cells were cultured in a stationary Phoenix for up to 5 days to assess cell viability and proliferation. The results show there is no significant difference in cell proliferation (∼5X growth on day 5) or viability (>90% viability on all days) when cultured in PhoenixTM and compared to a standard 5% CO2 incubator. Similarly, SH-SY5Y cells were evaluated following ground (1-3 days) and air (30 min) shipments to understand the impact of transit vibrations on the cell cultures. The results indicate that there is no significant difference in SH-SY5Y cell proliferation (∼2X growth on day 3) or viability (>90% viability for all samples) when the cells are subjected to the vibrations of ground and air transportation when compared to control samples in a standard, stationary 5% CO2 incubator. Furthermore, the temperature, pressure, humidity, and accelerometer sensors log data during culture shipment to ensure that the sensitive ATMPs are handled with the appropriate care during transportation. The PhoenixTM technology innovation will significantly increase the accessibility, reproducibility, and quality-controlled transport of living ATMPs to benefit the widespread commercialization of ATMPs globally. These results demonstrate that PhoenixTM can transport sensitive cell lines with the same care as traditional culture techniques in a stationary CO2 incubator with higher yield, less time and labor, and greater quality control than frozen samples.

Azelnidipine Attenuates the Oxidative and NFκB Pathways in Amyloid-ß-Stimulated Cerebral Endothelial Cells.

Cerebral amyloid angiopathy (CAA), a condition depicting cerebrovascular accumulation of amyloid ß-peptide (Aß), is a common pathological manifestation in Alzheimer's disease (AD). In this study, we investigated the effects of Azelnidipine (ALP), a dihydropyridine calcium channel blocker known for its treatment of hypertension, on oligomeric Aß (oAß)-induced calcium influx and its downstream pathway in immortalized mouse cerebral endothelial cells (bEND3). We found that ALP attenuated oAß-induced calcium influx, superoxide anion production, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and calcium-dependent cytosolic phospholipase A2 (cPLA2). Both ALP and cPLA2 inhibitor, methylarachidonyl fluorophosphate (MAFP), suppressed oAß-induced translocation of NFκB p65 subunit to nuclei, suggesting that cPLA2 activation and calcium influx are essential for oAß-induced NFκB activation. In sum, our results suggest that calcium channel blocker could be a potential therapeutic strategy for suppressing oxidative stress and inflammatory responses in Aß-stimulated microvasculature in AD.

Cytosolic Phospholipase A2 Facilitates Oligomeric Amyloid-ß Peptide Association with Microglia via Regulation of Membrane-Cytoskeleton Connectivity.

Cytosolic phospholipase A2 (cPLA2) mediates oligomeric amyloid-ß peptide (oAß)-induced oxidative and inflammatory responses in glial cells. Increased activity of cPLA2 has been implicated in the neuropathology of Alzheimer's disease (AD), suggesting that cPLA2 regulation of oAß-induced microglial activation may play a role in the AD pathology. We demonstrate that LPS, IFNγ, and oAß increased phosphorylated cPLA2 (p-cPLA2) in immortalized mouse microglia (BV2). Aß association with primary rat microglia and BV2 cells, possibly via membrane-binding and/or intracellular deposition, presumably indicative of microglia-mediated clearance of the peptide, was reduced by inhibition of cPLA2. However, cPLA2 inhibition did not affect the depletion of this associated Aß when cells were washed and incubated in a fresh medium after oAß treatment. Since the depletion was abrogated by NH4Cl, a lysosomal inhibitor, these results suggested that cPLA2 was not involved in the degradation of the associated Aß. To further dissect the effects of cPLA2 on microglia cell membranes, atomic force microscopy (AFM) was used to determine endocytic activity. The force for membrane tether formation (Fmtf) is a measure of membrane-cytoskeleton connectivity and represents a mechanical barrier to endocytic vesicle formation. Inhibition of cPLA2 increased Fmtf in both unstimulated BV2 cells and cells stimulated with LPS + IFNγ. Thus, increasing p-cPLA2 would decrease Fmtf, thereby increasing endocytosis. These results suggest a role of cPLA2 activation in facilitating oAß endocytosis by microglial cells through regulation of the membrane-cytoskeleton connectivity.

Evolution of the amyloid fiber over multiple length scales.

The amyloid is a natural self-assembled peptide material comparable in specific stiffness to spider silk and steel. Throughout the literature there are many studies of the nanometer-sized amyloid fibril; however, peptide mixtures are capable of self-assembling beyond the nanometer scale into micrometer-sized fibers. Here, atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to observe the self-assembly of the peptide mixtures in solution for 20 days and the fibers upon drying. Beyond the nanometer scale, self-assembling fibers differentiate into two morphologies, cylindrical or rectangular cross-section, depending on peptide properties. Microscopic observations delineate a four stage self-assembly mechanism: (1) protofibril (2-4 nm high and 15-30 nm wide) formation; (2) protofibril aggregation into fibrils 6-10 nm high and 60-120 nm wide; (3) fibril aggregation into large fibrils and morphological differentiation where large fibrils begin to resemble the final fiber morphology of cylinders (WG peptides) or tapes (Gd:My peptides). WG large fibrils are 50 nm high and 480 nm wide and Gd:My large fibrils are 10 nm high and 150 nm wide; (4) micrometer-sized fiber formation upon drying at 480 h resulting in 18.0 µm diameter cylindrical fibers (WG peptides) and 14.0 µm wide and 6.0 µm thick flat tapes (Gd:My peptides). Evolution of the large fiber morphology can be rationalized on the basis of the peptide properties.

Characterization of large amyloid fibers and tapes with Fourier transform infrared (FT-IR) and Raman spectroscopy.

Amyloids are self-assembled protein structures implicated in a host of neurodegenerative diseases. Organisms can also produce "functional amyloids" to perpetuate life, and these materials serve as models for robust biomaterials. Amyloids are typically studied using fluorescent dyes, Fourier transform infrared (FT-IR), or Raman spectroscopy analysis of the protein amide I region, and X-ray diffraction (XRD) because the self-assembled ß-sheet secondary structure of the amyloid can be easily identified with these techniques. Here, FT-IR and Raman spectroscopy analyses are described to characterize amyloid structures beyond just identification of the ß-sheet structure. It has been shown that peptide mixtures can self-assemble into nanometer-sized amyloid structures that then continue to self-assemble to the micrometer scale. The resulting structures are flat tapes of low rigidity or cylinders of high rigidity depending on the peptides in the mixture. By monitoring the aggregation of peptides in solution using FT-IR spectroscopy, it is possible to identify specific amino acids implicated in ß-sheet formation and higher order self-assembly. It is also possible to predict the final tape or cylinder morphology and gain insight into the structure's physical properties based on observed intermolecular interactions during the self-assembly process. Tapes and cylinders are shown to both have a similar core self-assembled ß-sheet structure. Soft tapes also have weak hydrophobic interactions between alanine, isoleucine, leucine, and valine that facilitate self-assembly. Rigid cylinders have similar hydrophobic interactions that facilitate self-assembly and also have extensive hydrogen bonding between glutamines. Raman spectroscopy performed on the dried tapes and fibers shows the persistence of these interactions. The spectroscopic analyses described could be generalized to other self-assembling amyloid systems to explain property and morphological differences.