Biology subjects

Ruggeri, F. S.

Publications and source records attributed to Ruggeri, F. S..

15 recordsLinked to original sources

bioRxiv2024

Computational redesign of TALE proteins for DNA-templated assembly of protein fibers

Many viral proteins self-assemble into capsid structures, often using their genetic material as a template for assembly. To date, de novo designed capsid-like proteins do not require genetic material as a template for assembly, which can be both an advantage and a disadvantage depending on the use case. Templates are indispensable, for example, in the assembly of linear structures with well-defined lengths. As a first step towards fully de novo designed templated assembly, here we redesign proteins from the Transcription activator-like effector (TALE) family of transcriptional regulators to polymerize on double-stranded DNA (dsDNA) templates. Starting from natural TALE protein sequences, we created idealized repeat proteins with sequence-independent DNA binding properties that cooperatively self-assemble to form linear protein-DNA complexes with template-controlled lengths. We used high-resolution atomic force microscopy (AFM) and cryo electron microscopy (cryo-EM) to characterize the three-dimensional structures of the DNA-protein hybrid complexes. In these structures, a protein filament helically wraps around the dsDNA using a binding mode similar to that of natural TALE proteins. As an example application of these materials, we show the system can be used for repetitive peptide antigen display at precisely controlled repeat distances, and that such immunogens elicit robust antigen-specific antibodies in mice.

biochemistry↗

bioRxiv2023

Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates

In addition to the native state, proteins can form liquid-like condensates, viscoelastic condensates, such as gels, as well as solid-like condensates, such as amyloid fibrils, crystals and amorphous materials. The material properties of these condensates play important roles in their cellular functions, with aberrant liquid-to-solid phase transitions having been implicated in neurodegenerative diseases. However, the molecular changes and resultant material properties across the whole phase space of condensates are complex and yet to be fully understood. The extreme sensitivity to their environment, which enables their biological function, is also what makes protein condensates particularly challenging experimental targets. Here, we provide a characterisation of the ageing behaviour of the full-length fused in sarcoma (FUS) protein. We achieve this goal by using a microfluidic sample deposition technology to enable the application of surface-based techniques to the study of biological condensates. We first demonstrate that we maintain relevant structural features of condensates in physiologically-relevant conditions on surfaces. Then, using a combination of atomic force microscopy and vibrational spectroscopy, we characterise the spatio-temporal changes in the structure and mechanical properties of the condensates to reveal local phase transitions in individual condensates. We observe that initially dynamic, fluid-like condensates undergo a global increase in elastic response conferred by an increase in the density of cation-{pi} intermolecular interactions. Solid-like structures form first at condensate-solvent interfaces, before heterogeneously propagating throughout the aged fluid core. These solid structures are composed of heterogenous, non-amyloid {beta}-sheets, which are stabilised by hydrogen-bonding interactions not observed in the fluid state. Overall, this study identifies the molecular conformations associated with different physical states of FUS condensates, establishing a technology platform to understand the role of phase behaviour in condensate function and dysfunction.

biophysics↗

bioRxiv2023

Maturation-dependent changes in the structure and seeding capacity of Aβ42 amyloid fibrils

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-{beta} network of hydrogen bonds. This process underlies a variety of human diseases, and can be exploited to develop versatile functional biomaterials. Thus, amyloid aggregation has been widely studied, shedding light on the properties of fibrils and their intermediates. A question that remains open concerns the microscopic processes that underlie the long-time behaviour of the fibrillar assemblies. Here, we use atomic force microscopy to observe that the fibrils undergo a maturation process, with an increase in both fibril length and thickness, and a change in the cross-{beta} sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates through secondary nucleation. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein based materials.

biophysics↗

bioRxiv2023

ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/533832v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@16f3e10org.highwire.dtl.DTLVardef@538f39org.highwire.dtl.DTLVardef@1e00d4eorg.highwire.dtl.DTLVardef@89967d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

A multiplex platform to identify mechanisms and modulators of proteotoxicity in neurodegeneration

Neurodegenerative disorders are a family of diseases that remain poorly treated despite their growing global health burden. A shared feature of many neurodegenerative disorders is the accumulation of toxic misfolded proteins. To gain insight into the mechanisms and modulators of protein misfolding, we developed a multiplex reverse genetics platform. Using this novel platform 29 cell-based models expressing proteins that undergo misfolding in neurodegeneration were probed against more than a thousand genetic modifiers. The resulting data provide insight into the nature of modifiers that act on multiple misfolded proteins as compared to those that show activity on only one. To illustrate the utility of this platform, we extensively characterized a potent hit from our screens, the human chaperone DNAJB6. We show that DNAJB6 is a general modifier of the toxicity and solubility of multiple amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD)-linked RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1. Biophysical examination of DNAJB6 demonstrated that it co-phase separates with, and alters the behavior of FUS containing condensates by locking them into a loose gel-like state which prevents their fibrilization. Domain mapping and a deep mutational scan of DNAJB6 support the critical importance for DNAJB6 phase separation in its effects on multiple RNA-binding proteins. Crucially, these studies also suggest that this property can be further tuned to generate novel variants with enhanced activity that might illuminate potential avenues for clinical translation.

neuroscience↗

bioRxiv2022

Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains

Neurodegenerative diseases, such as Alzheimers Disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low molecular weight aggregates play a key role in disease-associated toxicity. Within these aggregates, protofibrillar loop-like structures have been observed for a variety of amyloid systems and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils has largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterise amyloid ring structures derived from the brains of AD patients. We analyse the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. Furthermore, we show that these findings can be extended to several amyloid systems, giving a general framework relating the mechanical properties of assemblies and the conditions in which they can form loop structures. These results explain the diversity in the structures formed from protein aggregation and sheds light on the links between early forms of flexible ring-forming aggregates and their role in disease.

molecular biology↗

bioRxiv2022

Solid/liquid coexistence during aging of FUS condensates

A wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.

biophysics↗

bioRxiv2022

Enhanced surface nano-analytics of transient biomolecular processes

The study of the physical and chemical properties of biomolecules enables the characterisation of fundamental molecular processes and mechanisms in health and disease. Bulk and single-molecule analytical methods provide rich information on biomolecules, but often require high concentrations and sample preparation away from physiologically relevant conditions. Here, we present the development and application of a lab-on-a-chip approach which combines rapid sample preparation, mixing and deposition to integrate with a range of nano-analytical methods in chemistry and biology, providing enhanced sensitivity and single molecule resolution. We demonstrate that this method empowers multidimensional study of heterogenous biomolecular systems in physiological buffers and concentrations over multiple length scales by nanoscopy and vibrational spectroscopy. We illustrate the capabilities of this platform by capturing and analysing the structural conformations of transient oligomeric species formed at the early stages of the self-assembly of -synuclein, which are associated with the onset of Parkinsons disease. TEASERMaintaining the heterogeneity and structural integrity of monomers and oligomers enables their quantitative study.

biophysics↗

bioRxiv2021

Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties, and neurotoxicity of mutant Httex1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays an important role in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity. HighlightsO_LIThe Nt17 and polyQ domains synergistically promote Httex1 aggregation. C_LIO_LIThe Nt17 domain is a key determinant of the lateral association and morphology of fibrils. C_LIO_LIThe Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1. C_LIO_LINt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake. C_LIO_LINt17 serves as one of the master regulators of Httex1 aggregation, cellular uptake and toxicity. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/431207v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@7afd68org.highwire.dtl.DTLVardef@92c67aorg.highwire.dtl.DTLVardef@24823dorg.highwire.dtl.DTLVardef@e2a835_HPS_FORMAT_FIGEXP M_FIG The Nt17 domain: A master switch of Httex1 aggregation, uptake, subcellular localization and neurotoxicity. In this paper, we showed that 1) the Nt17 and polyQ domains synergistically promote Httex1 aggregation; 2) the Nt17 domain is a key determinant of the lateral association and morphology of fibrils in vitro, 3) Nt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake in primary neurons; 4) the Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1 in primary neurons. The figure was created with Biorender and https://www.vectorstock.com/royalty-free-vector/icon-on-and-off-toggle-switch-button-white-design-vector-30148026 C_FIG

neuroscience↗

bioRxiv2020

The Hsc70 Disaggregation Machinery RemovesMonomer Units Directly from α-Synuclein FibrilEnds

Molecular chaperones contribute to the maintenance of cellular protein homeostasis through a wide range of mechanisms, including the assistance of de novo protein folding, the rescue of misfolded proteins, and the prevention of amyloid formation. Chaperones of the Hsp70 family have a striking capability of disaggregating otherwise irreversible aggregate structures such as amyloid fibrils that accumulate during the development of neurodegenerative diseases. However, the mechanisms of this key emerging functionality remain largely unknown. Here, we bring together microfluidic measurements with kinetic analysis and show that that the Hsp70 protein heat chock complement Hsc70 together with its two co-chaperones DnaJB1 and the nucleotide exchange factor Apg2 is able to completely reverse the aggregation process of alpha-synuclein, associated with Parkinsons disease, back to its soluble monomeric state. Moreover, we show that this reaction proceeds with first order kinetics in a process where monomer units are taken off directly from the fibril ends. Our results demonstrate that all components of the chaperone triad are essential for fibril disaggregation. Lastly, we quantify the interactions between the three chaperones as well as between the chaperones and the fibrils in solution, yielding both binding stoichiometries and dissociation constants. Crucially, we find that the stoichiometry of Hsc70 binding to fibrils suggests Hsc70 clustering at the fibril ends. Taken together, our results show that the mechanism of action of the Hsc70-DnaJB1-Apg2 chaperone system in disaggregating -synuclein fibrils involves the removal of monomer units without any intermediate fragmentation steps. These findings are fundamental to our understanding of the suppression of amyloid proliferation early in life and the natural clearance mechanisms of fibrillar deposits in Parkinsons disease, and inform on the possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies and related neurodegenerative diseases.

biophysics↗

bioRxiv2020

Infrared Nanospectroscopy Reveals the Molecular Interaction Fingerprint of an Aggregation Inhibitor with Single Aβ42 Oligomers

Very significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimers and Parkinsons diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule level morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the interaction between single A{beta}42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, originally an anticancer drug capable recently shown to be able to inhibit A{beta}42 aggregation in animal models of Alzheimers disease. Our results demonstrate that the carbonyl group of this compound interacts with A{beta}42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as powerful tool in structure-based drug discovery for protein misfolding diseases.

biophysics↗

bioRxiv2020

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.

biophysics↗

bioRxiv2019

Small molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer’s disease

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that has been shown to modify the properties of their monomeric states. Here, we identify a small molecule capable of binding and sequestering the amyloid-{beta} peptide (A{beta}) in its monomeric, soluble state. Our analysis reveals that this compound interacts with A{beta} and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterise this interaction using biophysical experiments and integrative structural ensemble determination methods. We thus observe that this small molecule has the remarkable effect of increasing the conformational entropy of monomeric A{beta} while decreasing its hydrophobic surface area. We then show that this small molecule rescues a Caenorhabditis elegans model of A{beta}-associated toxicity in a manner consistent with the mechanism of action identified from the in silico and in vitro studies. These results provide an illustration of the strategy of targeting the monomeric states of disordered proteins with small molecules to alter their behaviour for therapeutic purposes.

biophysics↗

bioRxiv2019

ADSoluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression

Soluble aggregates of amyloid-{beta} (A{beta}) have been associated with neuronal and synaptic loss in Alzheimers disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we studied the soluble A{beta} aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls. We found that these aggregates vary structurally and in their mechanisms of toxicity. More small aggregates of A{beta} that can cause membrane permeabilization already found in MCI; in established AD, the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells. These results suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

neuroscience↗

bioRxiv2018

N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting.

Phosphorylation of exon1 of the Huntingtin protein (Httex1) has been shown to play important roles in regulating the structure, toxicity and cellular properties of N-terminal fragments and the full-length Huntingtin protein. Here, we investigated and compared the effect of bona fide phosphorylation at S13 and/or S16 on the structure, aggregation, membrane binding, and subcellular properties of mutant Httex1-Q18A. We show that serine phosphorylation at either S13 or S16 strongly disrupts the amphipathic -helix of the N-terminus, inhibits the aggregation of mutant Httex1 and prompts the internalization and nuclear targeting of Httex1 preformed aggregates. In synthetic peptides phosphorylation at S13 and/or S16 strongly disrupted the amphipathic -helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Our studies on peptides bearing a different combination of phosphorylation sites within Nt17 revealed a novel phosphorylation-dependent switch for regulating the structure of Httex1 involving crosstalk between phosphorylation at T3 and S13 or S16. Together, our results provide novel insights into the role of phosphorylation in regulating Httex1 structure and function in health and disease and underscore the critical importance of identifying enzymes responsible for regulating Htt phosphorylation and their potential as therapeutic targets for the treatment of Huntingtons disease.

neuroscience↗