Biology subjects

van der Wel, P. C. A.

Publications and source records attributed to van der Wel, P. C. A..

8 recordsLinked to original sources

bioRxiv2025

Probing How Anti-Huntingtin Antibodies Bind The Fibrillar Fuzzy Coat Using Solid-State NMR

Antibodies are critical for the immune response and serve as important tools due to their ability to recognize specific amino acid sequences, or epitopes. Based on the latter, they are utilized as diagnostic tools in biological and biomedical research. Huntingtons disease (HD) is a neurodegenerative condition caused by CAG repeat expansions in the huntingtin (HTT) gene and characterized by amyloid-like protein deposits in patients. Multiple anti-HTT antibodies are used in HD research for their ability to recognize specific HTT inclusions in both post-mortem tissue and in laboratory conditions. Some of the antibodies are seen as detectors of distinct structural motifs. However, most knowledge of their binding mechanism stems from studies of soluble monomers or short fragments of the epitopes, rather than the aggregated, misfolded target protein. Here, we investigate how MW8 antibodies interact with HTT exon 1 (HTTex1) fibrils, using solid-state NMR, electron microscopy, and complementary techniques. Magic angle spinning NMR revealed localized impacts of the antibody on exposed parts of the HTTex1 fibrils: the flanking segments that form its fuzzy coat. Antibody binding affected the structure and dynamics of the fuzzy coat, but also modulated the propensity for forming supramolecular fibril clusters, which has important implications for (reducing) cytotoxicity.

biochemistry↗

bioRxiv2024

Direct observation of secondary nucleation in huntingtin amyloid formation by High-Speed Atomic Force Microscopy

Amyloid fibril formation is a hallmark of various neurodegenerative diseases such as Huntingtons (HD), Alzheimers and Parkinsons disease. The protein aggregation process involves slow nucleation events followed by rapid growth and elongation of formed fibrils. Understanding the pathways of amyloid formation is key to development of novel therapeutic agents that can interfere with the pathogenic protein misfolding events. Recent studies of aggregation by polypeptides from Alzheimers and Huntingtons disease have identified the importance of a poorly understood secondary nucleation process that may even be the dominant source of protein aggregate formation. Here we focus on the polyglutamine-expansion disorder HD and employ mechanistic and structural studies to study different aspects of secondary nucleation in the aggregation of huntingtin Exon 1 (HttEx1). Notably, we apply high-speed atomic force microscopy (HS-AFM) to directly observe the process on the single-particle level and in real time. Our observations show unique features of the amyloid formation dynamics in real time, including secondary nucleation, elongation and the formation of large bundles of fibrils as a result of nucleated branching. We examine the role of HttEx1 flanking segments during the aggregation process, revealing that the N-terminal HttNT segment exhibits a clear primary nucleation-aggregation-enhancing ability, however, that it does not seem to induce or affect the secondary nucleation process. The obtained results illuminate the complex aggregation process of HttEx1 and have implications for attempts to inhibit or modulate it for therapeutic purposes.

molecular biology↗

bioRxiv2024

Resolving atomic-level dynamics and interactions of high-molecular weight hyaluronic acid in biomimetic ECM hydrogels by multidimensional solid-state NMR

High molecular weight (HMW) hyaluronic acid (HA) is a highly abundant natural polysaccharide and a fundamental component of the extracellular matrix (ECM). Its size and concentration regulate tissues macro- and microenvironments, and its upregulation is a hallmark feature of certain tumors. Yet, the conformational dynamics of HMW-HA and how it engages with components of the ECM microenvironment remain poorly understood on the molecular level. Probing the molecular structure and dynamics of HMW polysaccharides in a hydrated, physiological-like environment is crucial but also technically challenging. Here, we deploy advanced magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy in combination with isotopic enrichment to enable an in-depth study of HMW-HA to address this challenge. This approach resolves multiple coexisting HA conformations and dynamics as a function of environmental conditions. By combining 13C-labeled HA with unlabeled ECM components, we detect by MAS NMR HA-specific changes in global and local conformational dynamics as a consequence of hydration and ECM interactions. These measurements reveal atom-specific variations in dynamics and structure of the N-acetylglucosamine (GlcNAc) moiety of HA. We discuss possible implications for interactions that stabilize the structure of HMW-HA and facilitate its recognition by HA-binding proteins. The described methods apply similarly to studies of the molecular structure and dynamics of HA in tumor contexts and in other biological tissues, as well as HMW-HA hydrogels and nanoparticles used for biomedical and/or pharmaceutical applications.

biophysics↗

bioRxiv2023

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils from Huntington's disease

Neurodegeneration in Huntingtons disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinsons and Alzheimers diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report new magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a new understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

molecular biology↗

bioRxiv2023

Inhibitor-based modulation of huntingtin aggregation reduces fibril toxicity

Huntingtons disease (HD) is a neurodegenerative disorder in which mutated fragments of the huntingtin protein (Htt) undergo misfolding and aggregation. Since misfolded and aggregated proteins can cause cellular stress and cytotoxicity, there is an interest in the development of small molecule aggregation inhibitors as potential modulators of HD pathogenesis. Here, we study how a polyphenol modulates the aggregation mechanism of huntingtin exon 1 (HttEx1) even at sub-stoichiometric ratios. Sub-stoichiometric amounts of curcumin impacted the primary and/or secondary nucleation events, extending the pre-aggregation lag phase. Remarkably, the disrupted aggregation process changed both the aggregate structure and its cell metabolic properties. When administered to neuronal cells, the break-through protein aggregates induced a significantly reduced cellular stress compared to aggregates formed in absence of inhibitors. Structural analysis by electron microscopy, small angle X-ray scattering (SAXS), and solid-state NMR spectroscopy identified changes in the fibril structures, probing both the flanking domains that form the fuzzy coat, as well as the fibril core. The biggest changes affected the latter, which we propose to relate to the presence or absence of polyglutamine (polyQ) {beta}-hairpin structures. Our findings highlight multifaceted consequences of small molecule inhibitors that modulate the protein misfolding landscape, with potential implications for treatment strategies in HD and other amyloid disorders.

biophysics↗

bioRxiv2023

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

biophysics↗

bioRxiv2022

Selective observation of semi-rigid residues on the surface of dynamically complex mutant huntingtin protein fibrils

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid {beta}-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant proteins expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of intermediate motion selection (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latters detection with specific dyes and positron emission tomography tracers. HighlightsO_LIMutant huntingtin exon 1 fibrils feature a broad range of molecular dynamics. C_LIO_LIMolecular motion is coupled to water dynamics outside the fiber core. C_LIO_LIDynamics-based spectral editing ssNMR reveals mobile non-core residues. C_LIO_LIIntermediate-motion selection via dipolar dephasing of rigid sites. C_LIO_LISemi-mobile glutamines outside the fiber core observed and identified. C_LI

biophysics↗

bioRxiv2021

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

biophysics↗