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).

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.

Unlabelled: 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.

Ordered and Disordered Segments of Amyloid-β 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 (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9).

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.

Fluorescence quenching by lipid encased nanoparticles shows that amyloid-β 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-β oligomers have a preferred molecular orientation.

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.

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

We explored structural details of fibrils formed by a mutated amyloid β (Aβ(1-40)) peptide carrying a Phe to Lys mutation, which was shown to completely abolish the toxicity of the molecule. Computer models suggest that the positively charged Lys side chain is expelled from the hydrophobic fibril interior upon fibrillation. This can be accommodated by either a 180° flip of the entire lower β-strand (model M1) or local perturbations of the secondary structure in the direct vicinity of the mutated site (model M2).

Major Reaction Coordinates Linking Transient Amyloid-β 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 Aβ oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel β-sheet structure observed in mature fibrils.

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

There are three specific regions in the Amyloid beta (Aβ) 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 Zn and curcumin on Aβ, and compare these with their previously reported effects on Aβ.

Stereoisomers Probe Steric Zippers in Amyloid-β.

Shape complementarity between close-packed residues plays a critical role in the amyloid aggregation process. Here, we probe such "steric zipper" interactions in amyloid-β (Aβ), 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.

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

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

Small oligomers of the amyloid β (Aβ) 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.

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.