Major Reaction Coordinates Linking Transient Amyloid-ß Oligomers to Fibrils Measured at Atomic Level.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aß40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel ß-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two ß-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel ß-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aß40 fibrils and a focal point of mutations linked to early onset Alzheimer's disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel ß-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aß40 oligomers to fibrils.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

Significant structural differences between transient amyloid-ß oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer's mutations.

Small oligomers of the amyloidâ€…ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aß mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Dissolving Fusion Oncoprotein Condensates to Reverse Aberrant Gene Expression.

In a recent study, Wang and colleagues reported that a significant fraction of cancer-associated fusion proteins display a common structural topology, including an N-terminal phase separation-prone region (PS) from one parent protein and a C-terminal DNA-binding domain (DBD) from the other. This is reminiscent of the structural topology of transcription factors and led to the hypothesis that the PS-DBD fusions form aberrant transcriptional condensates through phase separation, which was supported through transcriptomic data analysis and cellular condensate assays. The authors developed a high-throughput screen based upon time-lapse, high-content imaging to identify 114 compounds that dissolved condensates formed by a chromatin-dissociated mutant of FUS::ERG (FUS::ERGmut). One of these compounds, LY2835219, was shown to dissolve FUS::ERGmut condensates by promoting lysosome formation and was also active against condensates formed by other PS-DBD fusions, including EWS::FLI1. Finally, condensate dissolution by LY2835219 was shown to reverse aberrant gene expression driven by EWS::FLI1, although how this compound specifically marshals lysosomes to target some PS-DBD fusions and not other condensate-forming proteins remains elusive. This work not only highlights likely roles for aberrant condensate formation in the oncogenic function of PS-DBD fusions, but also provides proof of principle for mechanistically unbiased screening to identify compounds that modulate fusion protein-driven condensates and their oncogenic functions.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

Charting the human disease condensate dysregulome.

Although it is understood that myriad proteins function in cells within biomolecular condensates, it is unclear how protein condensation is altered in human disease. In this issue of Developmental Cell, Banani et al. show that mutations in disease-associated proteins may map to phase-separation-prone regions and thereby alter condensate formation.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

NUP98 fusion oncoproteins (FO) are drivers in pediatric leukemias and many transform hematopoietic cells. Most NUP98 FOs harbor an intrinsically disordered region from NUP98 that is prone to liquid-liquid phase separation (LLPS) in vitro. A predominant class of NUP98 FOs, including NUP98-HOXA9 (NHA9), retains a DNA-binding homeodomain, whereas others harbor other types of DNA- or chromatin-binding domains. NUP98 FOs have long been known to form puncta, but long-standing questions are how nuclear puncta form and how they drive leukemogenesis. Here we studied NHA9 condensates and show that homotypic interactions and different types of heterotypic interactions are required to form nuclear puncta, which are associated with aberrant transcriptional activity and transformation of hematopoietic stem and progenitor cells. We also show that three additional leukemia-associated NUP98 FOs (NUP98-PRRX1, NUP98-KDM5A, and NUP98-LNP1) form nuclear puncta and transform hematopoietic cells. These findings indicate that LLPS is critical for leukemogenesis by NUP98 FOs. SIGNIFICANCE: We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

Ordered and Disordered Segments of Amyloid-ß Drive Sequential Steps of the Toxic Pathway.

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aß). Aß oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aß fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aß10-40 or Aß1-9, is toxic by itself. The ordered part (Aß10-40) is the major determinant of how Aß attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aß enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aß to the toxic pathway. Our findings correlate well with Aß sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity.

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates ("oligomers") of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic "oligomer fold" may not be universal.

Fluorescence quenching by lipid encased nanoparticles shows that amyloid-ß has a preferred orientation in the membrane.

Short range plasmonic fields around a nanoparticle can modulate fluorescence or Raman processes. In lipid encased nanoparticles, this can potentially measure the relative depths of different parts of a membrane protein from the surface. We employ this technique to discover that membrane inserted amyloid-ß oligomers have a preferred molecular orientation.

Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles.

Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells.

Stereoisomers Probe Steric Zippers in Amyloid-ß.

Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such "steric zipper" interactions in amyloid-ß (Aß40), whose aggregation is linked to Alzheimer's disease, by replacing natural residues by their stereoisomers. Such mutations are expected to specifically destabilize the shape sensitive "packing" interactions, which may potentially increase their solubility and change other properties. We study the stereomutants DF19 and DL34 and also the DA2/DF4/DH6/DS8 mutant of Aß40. F19-L34 is a critical contact in a tightly packed region of Aß, while residues 1-9 are known to be disordered. While both DF19 and DL34 slow down the kinetics of aggregation and form amyloid fibrils efficiently, only DL34 increases the final solubility. DF19 gives rise to additional off-pathway aggregation which results in large, kinetically stable aggregates, and has lower net solubility. DA2/DF4/DH6/DS8 does not have an effect on the kinetics or the solubility. Notably, both DF19 and DL34 oligomers have a significantly lower level of interactions with lipid vesicles and live cells. We conclude that stereoisomers can cause complex site dependent changes in amyloid properties, and provide an effective tool to determine the role of individual residues in shaping the packed interiors of amyloid aggregates.

Perturbation of the F19-L34 Contact in Amyloid ß (1-40) Fibrils Induces Only Local Structural Changes but Abolishes Cytotoxicity.

We explored structural details of fibrils formed by a mutated amyloid ß (Aß(1-40)) peptide carrying a Phe19 to Lys19 mutation, which was shown to completely abolish the toxicity of the molecule. Computer models suggest that the positively charged Lys19 side chain is expelled from the hydrophobic fibril interior upon fibrillation. This can be accommodated by either a 180° flip of the entire lower ß-strand (model M1) or local perturbations of the secondary structure in the direct vicinity of the mutated site (model M2). This is accompanied by the formation of a new salt bridge between Glu22 and Lys28 in model M1. Experimentally, a novel contact between Phe20 and Leu34 as well as the significant structural perturbation of residues 20-23 could be confirmed. However, the mutated fibrils do not show the formation of any salt bridges. This demonstrates that although morphologically very robust, local perturbations of the Aß(1-40) sequence lead to moderate structural alterations with tremendous impact on the physiological importance of these aggregates, which may suggest alternative strategies for the development of a remedy against Alzheimer's disease.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-ß Conformation.

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-ß40 (Aß40), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a ß-turn, flanked by two ß-sheet regions. We use solid-state NMR data to confirm the presence of the ß-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic Aß fibrils. Significantly, this allows a "porin" like ß-barrel structure, providing a structural basis for proposed mechanisms of Aß oligomer toxicity.