Amyloids are highly organized cross β-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. Here, we show that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross β-sheet-rich conformation. Thus, in contrast to the original association of amyloids with diseases, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.Cells transport newly synthesized secretory proteins and peptides in vesicles via the endoplasmic reticulum (ER) and Golgi for release into the extracellular space (1,2). Some secretory cells, such as neuroendocrine cells and exocrine cells, store secretory proteins and peptides for extended time periods in a highly concentrated form in membrane-enclosed electron-dense cores termed "secretory granules" (1,3,4), which are derived from the Golgi complex. The dense cores of these granules are made up of large, insoluble secretory protein and peptide aggregates that are formed by self-association (4-6). The granules are not amorphous, but possess a distinct molecular organization, possibly of crystalline structures (7) or large intermolecular aggregates (5,8).Amyloid fibrils are cross-β-sheet structures that are primarily associated with several neurodegenerative diseases including Alzheimer's disease. However, amyloid fibril formation also provides biologically functional entities termed functional amyloids (9) and are present in Escherichia coli (10), silkworm (11), fungi (12), and mammalian skin (13). The cross-β-sheet motif is composed of intermolecular β-sheets along the fibril axis with the β-strands aligned perpendicularly to the fibril axis. An amyloid-like structure of peptide and protein hormones in secretory granules could explain most of their properties.To address the question whether peptide and protein hormones are stored in secretory granules in an amyloid-like aggregation state, we first asked if a diverse set of peptide and protein hormones could form amyloids in vitro at granule-relevant pH 5.5. 42 peptide and protein hormones from multiple species and organs were selected randomly, some linear and some cyclic, with a variety of different three dimensional structures (Table S2). This set of hormones was assayed for a capacity to form amyloids by the amyloid-specific dyes thioflavin T (Thio T), congo red (CR), luminescent conjugated polyelectrolyte probes (LCP), by the conformational transition into β-sheet-rich structure measured by circular dichroism (CD), and by the presence of fibrils in electron microscopy (EM) images. Furthermore, x-ray fiber diffraction was measured for a subset of hormones (Table S1). Only 10 hormones out of the 42 showed significant formation of...
Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting: i) early non-thioflavinophilic protein assemblies of Aβ1-42 and insulin preceding the formation of amyloid fibrils and ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer’s diease brain tissue (Aβ plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.
The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.
Protein aggregation is associated with a wide range of diseases, and molecular probes that are able to detect a diversity of misfolded protein assemblies are of great importance. The identification of prefibrillar states preceding the formation of well-defined amyloid fibrils is of particular interest both because of their likely role in the mechanism of fibril formation and because of the growing awareness that these species are likely to play a critical role in the pathogenesis of protein deposition diseases. Herein, we explore the use of an anionic oligothiophene derivative, p-FTAA, for detection of prefibrillar protein aggregates during in vitro fibrillation of three different amyloidogenic proteins (insulin, lysozyme, and prion protein). p-FTAA generally detected prefibrillar protein aggregates that could not be detected by thioflavine T fluorescence and in addition showed high fluorescence when bound to mature fibrils. Second, the kinetics of protein aggregation or the formation of amyloid fibrils of insulin was not extensively influenced by the presence of various concentrations of p-FTAA. These results establish the use of p-FTAA as an additional tool for studying the process of protein aggregation.
A wide range of neurodegenerative diseases are characterized by the deposition of multiple protein aggregates. Ligands for molecular characterization and discrimination of these pathological hallmarks are thus important for understanding their potential role in pathogenesis as well as for clinical diagnosis of the disease. In this regard, luminescent conjugated oligothiophenes (LCOs) have proven useful for spectral discrimination of amyloid-beta (Aβ) and tau neurofibrillary tangles (NFTs), two of the pathological hallmarks associated with Alzheimer’s disease. Herein, the solvatochromism of a library of anionic pentameric thiophene-based ligands, as well as their ability to spectrally discriminate Aβ and tau aggregates, were investigated. Overall, the results from this study identified distinct solvatochromic and viscosity-dependent behavior of thiophene-based ligands that can be applied as indices to direct the chemical design of improved LCOs for spectral separation of Aβ and tau aggregates in brain tissue sections. The results also suggest that the observed spectral transitions of the ligands are due to their ability to conform by induced fit to specific microenvironments within the binding interface of each particular protein aggregate. We foresee that these findings might aid in the chemical design of thiophene-based ligands that are increasingly selective for distinct disease-associated protein aggregates.
The default mode network (DMN) is a group of anatomically separate regions in the brain found to have synchronized patterns of activation in functional magnetic resonance imaging (fMRI). Mentation associated with the DMN includes processes such as mind wandering, autobiographical memory, self-reflective thought, envisioning the future, and considering the perspective of others. Abnormalities in the DMN have been linked to symptom severity in a variety of mental disorders indicating that the DMN could be used as a biomarker for diagnosis. These correlations have also led to the use of DMN modulation as a biomarker for assessing pharmacological treatments. Concurrent research investigating the neural correlates of meditation, have associated DMN modulation with practice. Furthermore, meditative practice is increasingly understood to have a beneficial role in the treatment of mental disorders. Therefore we propose the use of DMN measures as a biomarker for monitoring the therapeutic effects of meditation practices in mental disorders. Recent findings support this perspective, and indicate the utility of DMN monitoring in understanding and developing meditative treatments for these debilitating conditions.
Molecular and electronic structures and optical absorption properties of oligothiophenes used for spectral assignment of amyloid deposits have been investigated for a family of probes known as luminescent conjugated oligothiophenes (LCOs). Theoretical absorption spectra have been determined using conformational averaging, combining classical molecular dynamics (MD) simulations with quantum mechanical/molecular mechanics (QM/MM) time-dependent density functional theory (TD-DFT) spectrum calculations. Theoretical absorption spectra are in excellent agreement with experiments, showing average errors below 5 nm for absorption maxima. To couple observed properties to molecular structures, a measure of planarity is defined, revealing a strong correlation between the transition wavelength of the first and dominating electronically excited state and dihedral rotations. It is shown that from this correlation, predictions can be made of the absorption properties of probes based only on information from MD trajectories. We show experimentally that red shifts observed in the excitation maxima of LCOs when bound to amyloid protein aggregates are also evident in absorption spectra. We predict that these red shifts are due to conformational restriction of the LCO in a protein binding pocket, causing a planarization of the conjugated backbone. On the basis of our studies of planarity, it is shown that such shifts are both possible and realistic.
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