Biology subjects

Andreasen, M.

Publications and source records attributed to Andreasen, M..

7 recordsLinked to original sources

bioRxiv2026

Structural and Mechanistic Basis of FapB Accessory Functional Amyloid Assembly in Pseudomonas

Functional amyloid formation in Pseudomonas biofilms is coordinated by the six-gene Fap operon (fapA-fapF), which governs the production, secretion, and assembly of extracellular fibrils that reinforce the biofilm matrix. FapC, FapB, and FapE constitute the extracellular amyloid. FapC is the major fibrillar component, whereas FapB and FapE occur at lower abundance and other accessory Fap proteins regulate fibril composition, processing, and secretion. We recently determined the structure of FapC, a cross-b-solenoid major Pseudomonas extracellular fibril. The assembly mechanism and architecture of FapB, however, have remained undefined. Here, we elucidate the structural and mechanistic basis of FapB accessory amyloid, from an intrinsically disordered monomer to a cross-b-solenoid fibril. Using well-defined monomeric preparations, controlled fibrillation assays, NMR spectroscopy, cryo-EM, and structure prediction, we show that FapB efficiently self-assembles into structurally homogeneous helical fibrils. FapB fibrillation is highly sensitive to its initial oligomeric state and purity, as well as to environmental factors such as pH. Monomeric FapC does not measurably perturb the disordered conformational ensemble of FapB, whereas preformed FapB and FapC fibrils reciprocally cross-seed each other, revealing direct kinetic coupling between the two amyloid components. Finally, we present a 3.4A resolution cryo-EM structure of the FapB accessory fibril, revealing a compact cross-b-solenoid architecture closely related to that of FapC. Together, these findings establish FapB as an independently amyloidogenic, structurally defined component of the Fap system, provide structural basis for the first of accessory cross-b-solenoid fibril, and reveal a mechanistic framework for the post-secretion assembly of multicomponent extracellular functional amyloids in Pseudomonas biofilms.

microbiology↗

bioRxiv2025

Structural defects in amyloid-β fibrils drive secondary nucleation

The nucleation of amyloid fibrils from monomeric protein, catalyzed by the surface of existing fibrils, is an important driver of many disorders such as Alzheimers and Parkinsons diseases. The structural basis of this secondary nucleation process, however, is poorly understood. Here, we ask whether secondary nucleation sites are found predominantly at rare growth defects: defects in the fibril core structure generated during their original assembly. We first demonstrate using the specific inhibitor of secondary nucleation, Brichos, that secondary nucleation sites on Alzheimers disease-associated fibrils composed of A{beta}40 and A{beta}42 peptides are rare compared to the number of protein molecules they contain. We then grow A{beta}40 fibrils under conditions designed to eliminate most growth defects while leaving the regular fibril morphology unchanged, and confirm the latter using cryo-electron microscopy. We measure both the ability of these annealed fibrils to promote secondary nucleation and the stoichiometry of their secondary nucleation sites, finding that both are greatly reduced as predicted. Re-analysis of published data for other proteins suggests that fibril growth defects that expose monomer planes or other structural units may also drive secondary nucleation generally, across most or all amyloids. These findings could unlock structure-based drug design of therapeutics that aim to halt amyloid disorders by inhibiting secondary nucleation sites.

biophysics↗

bioRxiv2025

Structural Basis of Pseudomonas FapC Biofilm-Forming Functional Amyloid Formation

Biofilm-protected Pseudomonas aeruginosa causes chronic infections that are difficult to treat. FapC, the major biofilm forming functional-amyloid in Pseudomonas, is essential for biofilm integrity, yet its structural details remain unresolved. Using an integrative structural biology approach, we combine solution NMR-based structural ensemble of unfolded monomeric FapC, a [~]3.3 [A] resolution CryoEM density map of FapC fibril, and all-atom MD simulations to capture transition from unfolded to folded monomer to fibrillar fold, providing a complete structural view of FapC biogenesis. CryoEM reveals a unique triple-layer {beta}-solenoid cross-{beta} fibril composed of a single protofilament. MD simulations initiated from monomeric and fibrillar FapC mapped structural transitions, offering mechanistic insights into amyloid assembly and disassembly. Understanding FapC reveals how Pseudomonas exploits functional amyloids for biofilm formation and establishes a structural and mechanistic foundation for developing therapeutics targeting biofilm-related infection and antimicrobial resistance.

microbiology↗

bioRxiv2023

Initial Steps of Chaperone-Aided Fibrillation of Pseudomonas aeruginosa Biofilm Forming Functional Amyloid FapC

Functional bacterial amyloids (FuBA) play a crucial role in the formation of biofilms, which are mediating chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution NMR spectroscopy, we show that both FapC and FapA, which are part of the same biofilm-forming protein operon, are intrinsically disordered proteins (IDPs) in their soluble monomeric state. These SSPs were determined and compared to the Alphafold models. We further demonstrate that the IDP chaperone FapA interacts with FapC and significantly slows down the formation of FapC fibrils, while maintaining the fibril morphology unchanged. Our NMR titration experiments reveal that [~]18% of the resonances show FapA induced chemical shift perturbations (CPSs) which has not been previously observed, the largest being for A82, N201, C237, C240, A241 and G245 residues. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the R1/L2 and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, [~]90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC. A temperature dependent effect of FapA was observed on FapC by ThT and NMR experiments. This study provides a detailed understanding of the interaction between the chaperone/chaperone-like FapA and the functional amyloid protein FapC, shedding light on the regulation and slowing down of amyloid formation. Our findings have important implications for the development of therapeutic strategies targeting biofilms and associated infections, leveraging these structural and mechanistic insights.

microbiology↗

bioRxiv2022

Uncovering the universality of self-replication in protein aggregation and its link to disease.

Fibrillar protein aggregates are a hallmark of the pathology of a range of human disorders, from prion diseases to dementias. Yet, the same aggregated structures that are formed in disease are also encountered in several functional contexts. The fundamental properties that determine whether these protein assembly processes are functional or, by contrast, pathological, have remained elusive. Here, we address this question by analysing the aggregation kinetics of a large set of self-assembling proteins, from those associated with disease, over those whose aggregates fulfil functional roles in biology, to those that aggregate only under artificial conditions. Remarkably, we find that essentially all systems that assemble by a nucleated-growth mechanism are capable of significant self-replication on experimentally accessible timescales. However, comparing the intrinsic timescales of self-replication with the timescales over which the corresponding aggregates form in a biological context yields a clear distinction; for aggregates which have evolved to fulfil a structural role, the rate of self-replication is too low to be significant on the biologically relevant timescale. By contrast, all analysed proteins that aggregate in the context of disease are able to self-replicate quickly compared to the timescale of the associated disease. Our findings establish the ability to self-replicate as both a ubiquitous property of protein aggregates and one that has the potential to be a key process across aggregation-related disorders.

biophysics↗

bioRxiv2021

Heparin promotes fibrillation of most phenol soluble modulin peptides from S. aureus: a possible strengthening of the bacterial biofilm

Phenol soluble modulins (PSMs) are virulence peptides secreted by different Staphylococcus aureus strains. In addition, PSMs are able to form amyloid fibrils which may strengthen the biofilm matrix. The highly sulfated glycosaminoglycan heparin promotes S.aureus infection but the basis for this is unclear. We hypothesized that heparin promotes PSM fibrillation and in this way aids bacterial colonization. Here we address this hypothesis using a combination of different biophysical techniques along with peptide microarrays. We find that heparin accelerates fibrillation of all -PSMs (except PSM2) and {delta}-toxin, but inhibits {beta}-PSMs fibrillation by blocking nucleation. Given that S. aureus secretes higher levels of -PSMs than {beta}-PSMs peptidess, heparin is likely to overall promote fibrillation. Heparin binding is driven by multiple positively charged lysine residues in -PSMs and {delta}-toxins, whose removal strongly reduces affinity. Binding of heparin does not alter the final fibril conformation. Rather, heparin provides a scaffold to catalyze or inhibit fibrillation. Our findings suggest that heparin may strengthen bacterial biofilm through increased PSM fibrillation. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/434294v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@99c3bdorg.highwire.dtl.DTLVardef@101d9b6org.highwire.dtl.DTLVardef@7c4058org.highwire.dtl.DTLVardef@1647eca_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Cross-talk between individual phenol soluble modulins in S. aureus biofilm formation

The infective ability of the opportunistic pathogen Staphylococcus aureus is associated with biofilm mediated resistance to host immune response and even disinfectants and indeed S. aureus is recognized as the most frequent cause of biofilm associated infections. Phenol-soluble modulin (PSM) peptides serve various roles in pathogenicity while also comprising the structural scaffold of S. aureus biofilms through self-assembly into functional amyloids, but the role of the individual PSMs during biofilm formation remains poorly understood and the molecular pathways of PSM self-assembly have proved challenging to identify. Here, we show a high degree of cooperation between individual PSMs during the formation of functional amyloids in biofilm formation. The fast aggregating PSM3 initiates the aggregation by forming unstable aggregates capable of seeding the formation of aggregates by other PSM peptides into the formation of stable amyloid structures. Using chemical kinetics along with spectroscopic techniques we dissect the molecular mechanism of aggregation of the individual peptides to show that PSM1, PSM3 and PSM{beta}1 display secondary nucleation whereas {beta}PSM2 aggregates through primary nucleation and elongation. Our findings suggest that the various PSMs have evolved to ensure fast and efficient biofilm formation through cooperation between individual peptides.

biophysics↗