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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Fluorescent Light‐up Probe for the Detection of Protein Aggregates

Abstract A fluorescent dye was decorated with water‐soluble pyridinium groups in order to be applied in the detection of cyclodextrins or DNA. The dye displays an enhancement of its emission intensity when the internal rotations are restricted due to the formation of an inclusion complex with cyclodextrins or upon interaction with DNA. In vivo , the fluorescent probe can stain protein aggregates with a selectivity comparable to the widely used Proteostat®.

Nunes da Silva, Raquel↗
DOE OSTI2019

Multivalent Polymer–Peptide Conjugates: A General Platform for Inhibiting Amyloid Beta Peptide Aggregation

Protein aggregation is implicated in multiple deposition diseases including Alzheimer's Disease, which features the formation of toxic aggregates of amyloid beta (Aβ) peptides. Many inhibitors have been developed to impede or reverse Aβ aggregation. Multivalent inhibitors, however, have been largely overlooked despite the promise of high inhibition efficiency endowed by the multivalent nature of Aβ aggregates. In this work, we report the success of multivalent polymer peptide conjugates (mPPCs) as a general class of inhibitors of the aggregation of Aβ 40 . Significantly delayed onset of fibril formation was realized using mPPCs prepared with three peptide/peptoid ligands covering a range of polymer molecular weights (MWs) and ligand loadings. Dose dependence studies showed that the nature of the ligands is a key factor in mPPC inhibition potency. The negatively charged ligand LPFFD leads to more efficient mPPCs compared to mPPCs with the neutral ligands, and is most effective at 7% ligand loading across different MWs. Molecular dynamics simulations along with dynamic light scattering experiments suggest that mPPCs form globular structures in solution due to ligand ligand interactions. Such interactions are key to the spatial proximity of ligands and thus to the multivalency effect of mPPC inhibition. Finally, excess ligand ligand interactions, however, reduce the accessibility of mPPC ligands to Aβ peptides, and impair the overall inhibition potency.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

De novo designed protein inhibitors of amyloid aggregation and seeding

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-β (Aβ) in Alzheimer’s disease (AD) and alpha-synuclein (αSyn) in Parkinson’s disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, “capping” them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aβ, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Cytoprotective role of ubiquitin against toxicity induced by polyglutamine-expanded aggregates

Highlights: • Formation of IBs composed of Q103 aggregates is independent of cellular Ub levels. • Ub deficiency is sufficient to cause the accumulation of Q103 aggregates in MEFs. • Ub deficiency leads to impaired autophagic clearance of Q103 aggregates in MEFs. • Ub-deficient MEFs are more susceptible to the toxicity caused by Q103 aggregates. Ubiquitin (Ub) homeostasis is important for cellular function and survival, especially under stress conditions. Recently, we have demonstrated that Ubc{sup −/−} (Ub-deficient) mouse embryonic fibroblasts (MEFs) exhibited reduced viability under oxidative stress induced by arsenite, which was not due to dysregulation of the antioxidant response pathway, but rather due to the potential toxicity caused by the misfolded protein aggregates. However, it is still not clear whether Ub deficiency is directly related to the accumulation of toxic protein aggregates, as arsenite itself triggers protein aggregation and renders cells into aberrant conditions such as reduced proteasome function and inhibition of autophagic flux. Therefore, under arsenite treatment, the outcome could be derived from the combination of multiple defective pathways. Furthermore, it has also been suggested that ubiquitination status of misfolded proteins may not be important for the formation of inclusion bodies composed of misfolded protein aggregates. We therefore wondered whether Ub deficiency is sufficient to trigger the accumulation of toxic protein aggregates inside the cells. In this study, we ectopically expressed polyQ-expanded aggregates (Q103) in MEFs and observed inclusion body formation at the juxtanuclear region, which was independent of cellular Ub levels. In contrast to arsenite treatment, polyQ expression did not affect proteasome function. However, we observed an increased accumulation of Q103 aggregates in Ubc{sup −/−} MEFs, which was due to impaired autophagic clearance. Finally, we demonstrated that the increased accumulation of Q103 aggregates under Ub deficiency dramatically reduced the viability of cells. Therefore, our results suggest that the maintenance of proper levels of cellular Ub is important to protect cells against the toxicity induced by the accumulation of protein aggregates.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Quantitative X-ray tomographic analysis reveals calcium precipitation in cataractogenesis

Cataracts, named for pathological light scattering in the lens, are known to be associated with increased large protein aggregates, disrupted protein phase separation, and/or osmotic imbalances in lens cells. We have applied synchrotron phase contrast X-ray micro-computed tomography to directly examine an age-related nuclear cataract model in Cx46 knockout (Cx46KO) mice. High-resolution 3D X-ray tomographic images reveal amorphous spots and strip-like dense matter precipitates in lens cores of all examined Cx46KO mice at different ages. The precipitates are predominantly accumulated in the anterior suture regions of lens cores, and they become longer and dense as mice age. Alizarin red staining data confirms the presence of calcium precipitates in lens cores of all Cx46KO mice. This study indicates that the spatial and temporal calcium precipitation is an age-related event associated with age-related nuclear cataract formation in Cx46KO mice, and further suggests that the loss of Cx46 promotes calcium precipitates in the lens core, which is a new mechanism that likely contributes to the pathological light scattering in this age-related cataract model.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Amyloidogenesis: What Do We Know So Far?

The study of protein aggregation, and amyloidosis in particular, has gained considerable interest in recent times. Several neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s (PD) show a characteristic buildup of proteinaceous aggregates in several organs, especially the brain. Despite the enormous upsurge in research articles in this arena, it would not be incorrect to say that we still lack a crystal-clear idea surrounding these notorious aggregates. In this review, we attempt to present a holistic picture on protein aggregation and amyloids in particular. Using a chronological order of discoveries, we present the case of amyloids right from the onset of their discovery, various biophysical techniques, including analysis of the structure, the mechanisms and kinetics of the formation of amyloids. We have discussed important questions on whether aggregation and amyloidosis are restricted to a subset of specific proteins or more broadly influenced by the biophysiochemical and cellular environment. The therapeutic strategies and the significant failure rate of drugs in clinical trials pertaining to these neurodegenerative diseases have been also discussed at length. At a time when the COVID-19 pandemic has hit the globe hard, the review also discusses the plausibility of the far-reaching consequences posed by the virus, such as triggering early onset of amyloidosis. Finally, the application(s) of amyloids as useful biomaterials has also been discussed briefly in this review.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

New Insights from Sum Frequency Generation Vibrational Spectroscopy into the Interactions of Islet Amyloid Polypeptides with Lipid Membranes

Studies of amyloid polypeptides on membrane surfaces have gained increasing attention in recent years. Several studies have revealed that membranes can catalyze protein aggregation and that the early products of amyloid aggregation can disrupt membrane integrity, increasing water permeability and inducing ion cytotoxicity. Nonetheless, probing aggregation of amyloid proteins on membrane surfaces is challenging. Surface-specific methods are required to discriminate contributions of aggregates at the membrane interface from those in the bulk phase and to characterize protein secondary structures in situ and in real time without the use of perturbing spectroscopic labels. Here, we review the most recent applications of sum frequency generation (SFG) vibrational spectroscopy applied in conjunction with computational modeling techniques, a joint experimental and computational methodology that has provided valuable insights into the aggregation of islet amyloid polypeptide (IAPP) on membrane surfaces. These applications show that SFG can provide detailed information about structures, kinetics, and orientation of IAPP during interfacial aggregation, relevant to the molecular mechanisms of type II diabetes. These recent advances demonstrate the promise of SFG as a new approach for studying amyloid diseases at the molecular level and for the rational drug design targeting early aggregation products on membrane surfaces.

Endocrinology & Metabolism↗
DOE OSTI2024

Effect of Aggregation and Molecular Size on the Ice Nucleation Efficiency of Proteins

Aerosol acts as ice-nucleating particles (INPs) by catalyzing the formation of ice crystals in clouds at temperatures above the homogeneous nucleation threshold (-38 °C). In this study, we show that the immersion mode ice nucleation efficiency of the environmentally relevant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), occurs at temperatures between -6.8 and -31.6 °C. Further, we suggest that this range is controlled by the RuBisCO concentration and protein aggregation. The warmest median nucleation temperature (-7.9 ± 0.8 °C) was associated with the highest concentration of RuBisCO (2 × 10 –1 mg mL –1 ) and large aggregates with a hydrodynamic diameter of ~10 3 nm. We investigated four additional chemically and structurally diverse proteins, plus the tripeptide glutathione, and found that each of them was a less effective INP than RuBisCO. Ice nucleation efficiency of the proteins was independent of the size (molecular weight) for the five proteins investigated in this study. In contrast to previous work, increasing the concentration and degree of aggregation did not universally increase ice nucleation efficiency. RuBisCO was the exception to this generalization, although the underlying molecular mechanism determining why aggregated RuBisCO is such an effective INP remains elusive.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Orientation and Conformation of Proteins at the Air–Water Interface Determined from Integrative Molecular Dynamics Simulations and Sum Frequency Generation Spectroscopy

Understanding the assembly of proteins at the air-water interface (AWI) informs the formation of protein films, emulsion properties, and protein aggregation. Determination of protein conformation and orientation at an interface is difficult to resolve with a single experimental or simulation technique alone. To date, the interfacial structure of even one of the most widely studied proteins, lysozyme, at the AWI remains unresolved. Here in this study, molecular dynamics (MD) simulations are used to determine if the protein adopts a side-on, head-on, or axial orientation at the AWI with two different forcefields, GROMOS-53a6 + SPC/E and a99SB-disp + TIP4P-D. Vibrational sum frequency generation (SFG) spectroscopy experiments and spectral SFG calculations validate consistency between the structure determined from MD and experiments. Overall, we show with strong agreement that lysozyme adopts an axial conformation at pH 7. Further, we provide molecular-level insight as to how pH influences the binding domains of lysozyme resulting in side-on adsorption near the isoelectric point of the lysozyme.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

ATP enhances at low concentrations but dissolves at high concentrations liquid-liquid phase separation (LLPS) of ALS/FTD-causing FUS

Highlights: • ATP is the energy currency but mysteriously with very high cellular concentrations. • ATP was recently decoded to dissolve LLPS of FUS whose aggregation leads to ALS. • Here we found for the first time that ATP can also enhance LLPS of FUS. • Therefore additional to being a hydrotrope, ATP also acts as a bivalent binder. • Our finding offers key clues in understanding age-dependent protein aggregation. ATP is the universal energy currency but mysteriously its cellular concentration is much higher than that needed for providing energy. Recently ATP was decoded to act as a hydrotrope to dissolve liquid-liquid phase separation (LLPS) of FUS whose aggregation leads to ALS/FTD. By DIC microscopy and NMR, here we characterized the effect of ATP on LLPS of FUS and its N-/C-terminal domains. Very unexpectedly, we found that like nucleic acids, ATP enhances LLPS of FUS at low but dissolves at high concentrations. Intriguingly, ATP monotonically dissolves LLPS of NTD, while it induces LLPS of CTD at low but dissolves at high concentrations. Our study reveals for the first time that ATP can enhance LLPS most likely by behaving as a bivalent binder. Most importantly, our results imply that age-dependent reduction of ATP concentrations may not only result in decreasing its capacity in preventing protein aggregation, but also in enhancing aggregation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS)

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Here, our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

TFEB-mediated activation of the lysosome-autophagy system affects the transduction efficiency of adeno-associated virus 2

Adeno-associated virus (AAV)-mediated gene transfer is an appealing therapeutic option due to AAV's safety profile. Effective delivery of AAV's genetic cargo to the nucleus, however, requires evasion of host cell barriers, including cellular clearance mechanisms mediated by the lysosome-autophagy system. We used AAV serotype 2 to monitor the autophagic response to cellular internalization of AAV and to characterize the effect of AAV-induced activation of autophagy on transgene expression. We found AAV2 internalization to induce activation of transcription factor EB, a master regulator of autophagy and lysosomal biogenesis, and upregulation of the lysosome-autophagy system. We showed that AAV2-induced activation of autophagy parallels a reduction in transgene expression, but also an increase in autophagic clearance of protein aggregates. These results can inform the design of AAV vectors with autophagy-modulating properties for applications ranging from the design of efficient gene delivery vectors to the treatment of diseases characterized by accumulation of autophagic cargo. - Highlights: • Cellular internalization of AAV2 results in activation of the transcription factor EB. • AAV2-induced autophagy activation parallels a reduction in AAV's transgene expression. • AAV2-induced autophagy activation enhances autophagic clearance of protein aggregates.

60 APPLIED LIFE SCIENCES↗