Magdalena I. Ivanova

Atomic structures of amyloid cross-β spines reveal varied steric zippers

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-β spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of β-sheets, with the facing side chains of the two sheets interdigitated in a dry ‘steric zipper’. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer’s amyloid-β and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, α-synuclein and β2-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.

Molecular basis for insulin fibril assembly.

In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio–dependent manner, suggesting that this segment is central to the cross-β spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 Å resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated β-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.

Structure-based design of functional amyloid materials.

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses.

Molecular Basis for Preventing α-Synuclein Aggregation by a Molecular Tweezer

Background: The molecular tweezer, CLR01, binds to Lys and prevents aggregation of α-synuclein. Results: CLR01 binds directly to monomeric α-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure. Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.

Molecular level insights into thermally induced α-chymotrypsinogen A amyloid aggregation mechanism and semiflexible protofibril morphology

Understanding nonnative protein aggregation is critical not only to a number of amyloidosis disorders but also for the development of effective and safe biopharmaceuticals. In a series of previous studies [Weiss et al. (2007) Biophys. J. 93, 4392-4403; Andrews et al. (2007) Biochemistry 46, 7558-7571; Andrews et al. (2008) Biochemistry 47, 2397-2403], α-chymotrypsinogen A (aCgn) and bovine granulocyte colony stimulating factor (bG-CSF) have been shown to exhibit the kinetic and morphological features of other nonnative aggregating proteins at low pH and ionic strength. In this study, we investigated the structural mechanism of aCgn aggregation. The resultant aCgn aggregates were found to be soluble and exhibited semiflexible filamentous aggregate morphology under transmission electron microscopy. In addition, the filamentous aggregates were demonstrated to possess amyloid characteristics by both Congo red binding and X-ray diffraction. Peptide level hydrogen exchange (HX) analysis suggested that a buried native β-sheet comprised of three peptide segments (39-46, 51-64, and 106-114) reorganizes into the cross-β amyloid core of aCgn aggregates and that at least ∼50% of the sequence adopts a disordered structure in the aggregates. Furthermore, the equimolar, bimodal HX labeling distribution observed for three reported peptides (65-102, 160-180, and 229-245) suggested a heterogeneous assembly of two molecular conformations in aCgn aggregates. This demonstrates that extended β-sheet interactions typical of the amyloid are sufficiently strong that a relatively small fraction of polypeptide sequence can drive formation of filamentous aggregates even under conditions favoring colloidal stability.

Distinct C9orf72-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity.

Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity.

Reduction of the C191-C220 disulfide of α-chymotrypsinogen A reduces nucleation barriers for aggregation

Proper disulfide formation can be essential for the conformational stability of natively folded proteins. For proteins that must unfold in order to aggregate, disruption of native disulfides may therefore promote aggregation. This study characterizes differences in the aggregation process for wild-type (WT) α-chymostrypsinogen A (aCgn) and the same molecule with one of its native disulfides (C191-C220) reduced to free thiols (aCgnSH) at acidic pH, where WT aCgn forms semi-flexible amyloid polymers. Loss of the disulfide leads to no discernable differences in folded monomer secondary or tertiary structure based on circular dichroism (CD) or intrinsic fluorescence (FL), and causes a small decrease in the free energy change upon unfolding. After unfolding-mediated aggregation, the resulting amyloid morphology and structure are similar or indistinguishable for aCgn and aCgnSH by CD, FL, ThT binding, multi-angle laser light scattering, and transmission electron microscopy. Aggregates of aCgn and aCgnSH are also able to cross-seed with monomers of the other species. However, aggregates of aCgnSH are more resistive than aCgn aggregates to urea-mediated dissociation, suggesting some degree of structural differences in the aggregated species that was not resolvable in detail without higher resolution methods. Mechanistic analyses of aggregation kinetics indicate that the initiation or nucleation of new aggregates from aCgnSH involves a mono-molecular rate limiting step, possibly the unfolding step. In contrast, that for aCgn involves an oligomeric intermediate, suggesting native disulfide linkages help to hinder non-native protein aggregation by providing conformational barriers to key nucleation event(s).

Erratum to: A Novel “Molecular Tweezer” Inhibitor of α-Synuclein Neurotoxicity In Vitro and In Vivo

Erratum to: Neurotherapeutics DOI 10.1007/s13311-012-0105-1 Rajeswari Lakshmanan was listed incorrectly on the original publication of this article. The author list is corrected here.

INVESTIGATING THE ROLE OF UBIQUILIN2 IN AGE-RELATED NEURODEGENERATION

Background:Abnormal cholesterol metabolism is suspected as one of the factors contributing to Alzheimer disease (AD) pathogenesis. We and others have previously shown that g-secretase dysfunction, which appears to be a main consequence caused by clinical presenilin mutations relevant to familial AD, increases cholesterol level in non-neuronal cells [1, 2]. Additionally, we proposed that increase of one of the g-secretase substrates, amyloid precursor protein Cterminal fragments (APP-CTFs), is a possible mediator of the cholesterol increase [2]. In this study, we examined the involvement of APP-CTFs in themetabolism of cholesterol and lipid droplets [3] in neuronally differentiated SH-SY5Y (nSY5Y) cells and in mouse embryonic fibroblasts lacking APP expression (MEFs-APPKO). Methods:nSY5Y cells differentiated by retinoic acid or MEFs-wild type (MEFs-WT) or MEFs-APPKOwere treated with a g-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). To suppress APP-CTF accumulation in nSY5Y cells upon DAPT treatment, cells were co-treated with inhibitors of a-secretase or b-secretase. Levels of lipid droplets and cholesterol were measured by oil red-O staining and enzymatic assay, respectively. Results:g-Secretase inhibition in nSY5Y cells by DAPT significantly increased levels of lipid droplet and cholesterol and affected the expression profile of the proteins involved in cholesterol metabolism, such as ABCA1, NPC1, sterol regulatory element-binding protein 2, and LDLR. Suppression of the DAPTinduced APP-CTFs accumulation completely rescued lipid droplet accumulation; however, cholesterol accumulation and abnormal expression profile of the proteins were not rescued by suppression of the APP-CTFs accumulation. Additionally, g-secretase inhibition induced lipid droplet accumulation only in MEFs-WT but not in MEFs-APPKO in contrast to cholesterol accumulation, which was detected in both of them upon DAPT treatment. Conclusions:These results indicate that g-secretase inhibition has complex effects on cellular lipid metabolism in neuronal and non-neuronal cells, partly involving accumulated APP-CTFs. References: 1) Grimm MO, et al. Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin. Nat Cell Biol. 2005;7(11):1118-1123. 2) Tamboli IY, et al. Loss of gamma-secretase function impairs endocytosis of lipoprotein particles and membrane cholesterol homeostasis. J Neurosci. 2008;28(46):1209712106. 3) Area-Gomez E, et al. Upregulated function of mitochondria-associated ER membranes in Alzheimer disease. EMBO J. 2012;31(21):4106-4123.