ABSTRACT
Apolipoprotein A‐I (apoA‐I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid‐free state, the apolipoprotein amphipathic a‐helices interact ...intra‐ and intermolecularly, providing structural stabilization by self‐association. We have reported that lipid‐free apoA‐I becomes amyloidogenic upon physiologically relevant (myeloperoxidase‐mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA‐I monomers and irreversibly disrupting selfassociation. The oxidized apoA‐I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA‐I. Of physiologic relevance, preformed oxidized apoA‐I amyloid fibrils induced amyloid formation in nonoxidized apoA‐I. This process was enhanced when self‐association of nonoxidized apoA‐I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA‐I were structurally similar to those formed by the oxidized protein, featuring a β‐structure‐rich amyloid fold alongside a‐helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA‐I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid‐free apoA‐I. Our results suggest that even partial Met oxidation of apoA‐I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL‐forming apoA‐I.—Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A‐I at the crossroads of HDL biogenesis and amyloid formation. FASEB J. 32, 3149–3165 (2018). www.fasebj.org
Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with ...disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.
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•Mutant huntingtin exon 1 displays hierarchical supramolecular polymorphism.•Fibril bundling is polymorph dependent, mediated by mobile segments on the surface.•N- and C-terminal flanking domains have distinct roles in the assembly mechanism.•Protein concentration is identified as a driver of huntingtin polymorphism.•Competing mechanisms provide a rationale for polymorphic behavior.
Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7 - 13 is ...one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13C and 15N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the “steric zipper” observed in the monoclinic crystals.
The aggregation of proteins and peptides into a variety of insoluble, and often non-native, aggregated states plays a central role in many devastating diseases. Analogous processes undermine the ...efficacy of polypeptide-based biological pharmaceuticals, but are also being leveraged in the design of biologically inspired self-assembling materials. This Trends article surveys the essential contributions made by recent solid-state NMR (ssNMR) studies to our understanding of the structural features of polypeptide aggregates, and how such findings are informing our thinking about the molecular mechanisms of misfolding and aggregation. A central focus is on disease-related amyloid fibrils and oligomers involved in neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease. SSNMR-enabled structural and dynamics-based findings are surveyed, along with a number of resulting emerging themes that appear common to different amyloidogenic proteins, such as their compact alternating short-β-strand/β-arc amyloid core architecture. Concepts, methods, future prospects and challenges are discussed.
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•The aggregation of peptides and proteins is a hallmark of many incurable diseases.•Solid-state NMR site-specifically probes the conformation of protein aggregates.•Solid-state NMR studies of dynamics and disordered regions yield unique insights.•Recent solid-state NMR structures point to common molecular themes and mechanisms.
Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about ...the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils.
Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances ...in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced
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C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.
DNP-enhanced correlation experiments acquired at 9.4 T and 100 K can yield site-specific structural data with remarkable sensitivity.
Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for ...biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.
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The peroxidation of cardiolipins by reactive oxygen species, which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular ...underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin (CL), while multidimensional solid-state NMR probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by affecting the dynamics of the cyt c N70-I85 Ω loop, which covers the heme cavity.
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•Cytochrome c is a cardiolipin-selective lipid peroxidase in intrinsic apoptosis•Lipidomics probe oxygenation products of cardiolipin peroxidation in vitro•Solid-state NMR shows folded, but dynamically perturbed, protein on lipid surface•PUFA lipid-coupled dynamics of 70–85 Ω loop regulate access to heme cavity
Li et al. show how cardiolipin acts as both a substrate and a dynamic regulator of cytochrome c's pro-apoptotic lipid peroxidase functionality. Combining solid-state NMR and mass spectrometry lipidomics, they find that localized motion of the 70–85 Ω loop regulates heme cavity access in the peroxidase-active peripherally membrane-bound protein.
Lead–tin (Pb–Sn) perovskites are a highly promising composition for single-junction and all-perovskite tandem solar cells due to their narrower bandgap and reduced toxicity. While the use of ...quasi-two-dimensional (quasi-2D) Ruddlesden–Popper phases has resulted in superior stability towards the environment and large improvement in the crystallization with respect to the 3D compositions, very little work has been done towards their deposition with scalable techniques. Here, PEA 2 (FA 0.5 MA 0.5 ) 4 (Pb 0.5 Sn 0.5 ) 5 I 16 ( n = 5) with a gradient structure is successfully prepared for the first time via a two-step blade coating. Perovskite films which are treated with tin( ii ) acetate (SnAc 2 ) along with N , N -dimethylselenourea (DMS) exhibit a reduced number of surface traps and enhanced surface crystallization, owing to the in situ formation of tin selenide (SnSe). Record devices with power conversion efficiency (PCE) of 15.06%, an open circuit voltage ( V OC ) of 0.855 V, and negligible hysteresis are obtained. More importantly, the hydrophobic SnSe significantly protects the active layer from the environment. These devices retain 91% of the original PCE after 10 days in ambient air (30–40% humidity) without encapsulation, and almost no degradation of the PCE is detected after over a month of storage in an inert atmosphere, and under continuous MPP tracking for 15 hours.
Several human diseases are associated with the formation of amyloid aggregates, but experimental characterization of these amyloid fibrils and their oligomeric precursors has remained challenging. ...Experimental and computational analysis of simpler model systems has therefore been necessary, for instance, on the peptide fragment GNNQQNY7−13 of yeast prion protein Sup35p. Expanding on a previous publication, we report here a detailed structural characterization of GNNQQNY fibrils using magic angle spinning (MAS) NMR. On the basis of additional chemical shift assignments we confirm the coexistence of three distinct peptide conformations within the fibrillar samples, as reflected in substantial chemical shift differences. Backbone torsion angle measurements indicate that the basic structure of these coexisting conformers is an extended β-sheet. We structurally characterize a previously identified localized distortion of the β-strand backbone specific to one of the conformers. Intermolecular contacts are consistent with each of the conformers being present in its own parallel and in-register sheet. Overall the MAS NMR data indicate a substantial difference between the structure of the fibrillar and crystalline forms of these peptides, with a clearly increased complexity in the GNNQQNY fibril structure. These experimental data can provide guidance for future work, both experimental and theoretical, and provide insights into the distinction between fibril growth and crystal formation.
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