Francesco Simone Ruggeri

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions

Summary Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

Nanoscale studies link amyloid maturity with polyglutamine diseases onset

The presence of expanded poly-glutamine (polyQ) repeats in proteins is directly linked to the pathogenesis of several neurodegenerative diseases, including Huntington’s disease. However, the molecular and structural basis underlying the increased toxicity of aggregates formed by proteins containing expanded polyQ repeats remain poorly understood, in part due to the size and morphological heterogeneity of the aggregates they form in vitro. To address this knowledge gap and technical limitations, we investigated the structural, mechanical and morphological properties of fibrillar aggregates at the single molecule and nanometer scale using the first exon of the Huntingtin protein as a model system (Exon1). Our findings demonstrate a direct correlation of the morphological and mechanical properties of Exon1 aggregates with their structural organization at the single aggregate and nanometric scale and provide novel insights into the molecular and structural basis of Huntingtin Exon1 aggregation and toxicity.

A critical concentration of N-terminal pyroglutamylated amyloid beta drives the misfolding of Ab1-42 into more toxic aggregates

A wide consensus based on robust experimental evidence indicates pyroglutamylated amyloid-β isoform (AβpE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimers disease. Furthermore, AβpE3-42 co-oligomerized with excess of Aβ1-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from Aβ1-42 alone. Here, we investigate quantitatively the influence of AβpE3-42 on biophysical properties and biological activity of Aβ1-42. We tested different ratios of AβpE3-42/Aβ1-42 mixtures finding a correlation between the biological activity and the structural conformation and morphology of the analyzed mixtures. We find that a mixture containing 5% AβpE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel β-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that AβpE3-42 causes template-induced misfolding of Aβ1-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on Aβ1-42 aggregation, above this threshold, the seed effect is not possible anymore and AβpE3-42 controls the total aggregation kinetics.

AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

Background A wide class of human diseases and neurodegenerative disorders, such as Alzheimer’s disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-β sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established. Methods: In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species. Conclusion: The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases.

Nanoplasmonic mid-infrared biosensor for in vitro protein secondary structure detection

Plasmonic nanoantennas offer new applications in mid-infrared (mid-IR) absorption spectroscopy with ultrasensitive detection of structural signatures of biomolecules, such as proteins, due to their strong resonant near-fields. The amide I fingerprint of a protein contains conformational information that is greatly important for understanding its function in health and disease. Here, we introduce a non-invasive, label-free mid-IR nanoantenna-array sensor for secondary structure identification of nanometer-thin protein layers in aqueous solution by resolving the content of plasmonically enhanced amide I signatures. We successfully detect random coil to cross β-sheet conformational changes associated with α-synuclein protein aggregation, a detrimental process in many neurodegenerative disorders. Notably, our experimental results demonstrate high conformational sensitivity by differentiating subtle secondary-structural variations in a native β-sheet protein monolayer from those of cross β-sheets, which are characteristic of pathological aggregates. Our nanoplasmonic biosensor is a highly promising and versatile tool for in vitro structural analysis of thin protein layers.

Silk micrococoons for protein stabilisation and molecular encapsulation.

Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules.

Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation‐Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs.

Micro- and nanoscale hierarchical structure of core-shell protein microgels

Protein nanofibrils were first discovered in the context of misfolding and neurodegenerative diseases but have recently been found in naturally occurring functional materials including algal adhesives, bacterial coatings, and even mammalian melanosomes. These physiologically beneficial roles have led to the exploration of their use as the basis for artificial protein-based functional materials for a range of applications as bioscaffolds and carrier agents. In this work, we fabricate core-shell protein microgels stabilized by protein fibrillation with hierarchical structuring on scales ranging from a few nanometers to tens of microns. With the aid of droplet microfluidics, we exploit fibrillar protein self-assembly together with the aqueous phase separation of a polysaccharide and polyethylene glycol to control the internal structure of the microgels on the micro- and nanoscales. We further elucidate the local composition, morphology, and structural characteristics of the microgels and demonstrate a potential application of core-shell protein microgels for controlling the storage and sequential release of small drug-like molecules. The controlled self-assembly of protein nanofibrils into hierarchical structures can be used in this manner to generate a class of nanomaterials with a range of potential functions and applications.

Determination of Polypeptide Conformation with Nanoscale Resolution in Water

The folding and acquisition of proteins native structure is central to all biological processes of life. By contrast, protein misfolding can lead to toxic amyloid aggregates formation, linked to the onset of neurodegenerative disorders. To shed light on the molecular basis of protein function and malfunction, it is crucial to access structural information on single protein assemblies and aggregates under native conditions. Yet, current conformation-sensitive spectroscopic methods lack the spatial resolution and sensitivity necessary for characterizing heterogeneous protein aggregates in solution. To overcome this limitation, here we use photothermal-induced resonance to demonstrate that it is possible to acquire nanoscale infrared spectra in water with high signal-to-noise ratio (SNR). Using this approach, we probe supramolecular aggregates of diphenylalanine, the core recognition module of the Alzheimers β-amyloid peptide, and its derivative Boc-diphenylalanine. We achieve nanoscale resolved IR spectra and maps in air and water with comparable SNR and lateral resolution, thus enabling accurate identification of the chemical and structural state of morphologically similar networks at the single aggregate ( i. e., fibril) level.

Nanobodies raised against monomeric ɑ-synuclein inhibit fibril formation and destabilize toxic oligomeric species

BackgroundThe aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS.ResultsWe show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity.ConclusionsThe results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.