Biology subjects

Bernfur, K.

Publications and source records attributed to Bernfur, K..

7 recordsLinked to original sources

bioRxiv2026

Errors in peptide synthesis are a source of discrepancies in Aβ42 studies

Amyloid-{beta}42 (A{beta}42) aggregation is highly sensitive to experimental conditions, making reproducibility a persistent challenge in Alzheimers disease research. Among the many variables that influence aggregation, the impact of peptide production remains poorly understood. Direct comparison of recombinant and chemically synthesised A{beta}42 prepared under carefully controlled conditions reveals that, despite following similar aggregation mechanism and forming the same predominant fibril structures, synthetic A{beta}42 aggregates more slowly and exhibits reduced seeding efficiency. Consequently, synthetic A{beta}42 produces fewer oligomeric species and displays lower cellular toxicity. Mass spectrometric analyses identify low-abundance sequence imperfections introduced during peptide synthesis as the origin of these differences. By linking synthesis-derived imperfections to variations in A{beta}42 behaviour, this work reveals a previously underappreciated source of discrepancies in amyloid studies. In addition, we provide a framework for evaluating the impact of sequence impurities on biophysical studies that are sensitive to peptide composition.

biochemistry↗

bioRxiv2025

On the transient interactions of α-synuclein in different dimensions

0.-Synuclein (Syn) is a neuronal protein predominantly found in the brain, whose native function seems to be associated with vesicle trafficking. While intrinsically disordered in solution, the first ca. 100 residues adopt an amphipathic -helical structure when the protein adsorbs onto membranes. Additionally, the aggregation of Syn into highly ordered {beta}-sheet rich amyloid fibrils is associated with Parkinso[n]s disease. The different regions of Syn and the interactions between them have been reported to play a key role in the behaviour of the protein in solution, its membrane binding, and its aggregation into fibrils. This study employs photo-induced cross-linking of unmodified proteins (PICUP) to capture and identify the transient contacts of Syn in different conformational states: free in solution, adsorbed to membranes, and aggregated into fibrils. By using tyrosine-to-phenylalanine mutations to block the reactivity of specific amino acid residues, we establish key cross-links in each state. In solution, we identify internal contacts between the N and C termini of monomers, as well as inter-monomer contacts between C termini in oligomers. When Syn is adsorbed to membranes, the internal cross-linking is blocked, while cross-linking between C-terminal regions persists. In fibrils, cross-linking is significantly reduced, primarily occurring between C-terminal residues of adjacent monomers. This work highlights the utility of PICUP for reporting on the transient contacts that occur on the pathways of self- and co-assembly of Syn.

biochemistry↗

bioRxiv2024

Tipping point in α-synuclein-membrane interactions: stable protein-covered vesicles or amyloid aggregation

-synuclein is a neuronal protein implicated in neurotransmitter release. Its function is thought to critically depend on the dynamic equilibrium between free and membrane-bound protein. -synuclein amyloid formation implicated in Parkinsons Disease was also shown to be modulated by lipid membranes. However, it remains elusive whether -synuclein-related pathology is due to loss-of-function or gain-of-toxic-function. To help address this question, we studied the coupling of the equilibrium between free and membrane-bound -synuclein and membrane-induced amyloid formation - phenomena that are usually treated separately. We present a description of the system on a wide range of length scales and timescales for lipid-to-protein ratio conditions where amyloid formation is either accelerated or inhibited by lipid membranes. We find a clear difference between the dynamics and heterogeneity of the protein-covered membrane interface in the two sets of conditions. In aggregation-accelerating conditions, the membrane interface is dynamic and heterogeneous with rapid exchange between free and membrane-bound protein, and disordered protein segments of varying lengths exposed to solution. All these characteristics of the membrane interface are likely to decrease the free energy barrier for amyloid formation. Conversely, the membrane interface is homogeneous and less dynamic in conditions where amyloid formation is inhibited. Importantly, any factors affecting the equilibrium between free and membrane-bound -synuclein may trigger a change from non-aggregating to aggregating conditions. Altogether, our results highlight a strong coupling of the dynamic equilibrium between the free and membrane-bound -synuclein and membrane-modulated amyloid formation and thus of the physiological function of -synuclein and its aberrant aggregation.

biochemistry↗

bioRxiv2024

Regulation of ADP-ribosyltransferase activity by ART domain dimerization in PARP15

PARP15 is a mono-ADP-ribosyltransferase with unknown functions. Its evolutionary relationship with PARP14 suggests roles in antiviral defense; its ability to modify RNA and localization to stress granules point to functions in the regulation of translation. PARP15 also modifies itself and other proteins using its ADP-ribosyltransferase (ART) domain and contains two macrodomains predicted to bind ADP-ribosyl on targets. We used biochemical and biophysical analysis to study how the ADP-ribosyltransferase activity of PARP15 is regulated. Here we show that the catalytic domain of PARP15 dimerizes with mid-nanomolar affinity, forming the same dimer interface in solution that had already been captured by X-ray crystallography of the domain. Furthermore, we show that the formation of dimers is a prerequisite for catalytic activity and that monomeric mutant variants of the domain were catalytically inactive. Our findings suggest a regulatory mechanism by which dimerization is linked to either target engagement or placement of a catalytic residue, rather than NAD+ co-substrate binding, and by which the two protomers of the dimer operate independent of one another. Together, our results uncover a novel mechanism of regulation in a PARP family enzyme, which might inspire new avenues of pharmacological intervention.

biochemistry↗

bioRxiv2021

The bacterial amyloids phenol soluble modulins from Staphylococcus aureus catalyze alpha-synuclein aggregation

Aggregated -synuclein (-syn) is the main constituent of Lewy bodies, the main pathological hallmark of Parkinsons disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric -syn. Braaks seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role that the microbiome might play in the pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called Phenol Soluble Modulins (PSMs). Here, we studied the kinetics of -syn aggregation under quiescent conditions in the presence or absence of four different PSM peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure -syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSM peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSM peptides displayed distinct effects on the kinetics of -syn aggregation. Kinetic analyses of the data suggest that while all four peptides catalyze -syn aggregation, the underlying mechanisms might differ with a model of nucleation and elongation fitting the -syn aggregation induced by PSM2 but not PSM1. The results of immunogold TEM imply that the aggregates are fibrillar and composed of -syn. Addition of the co-aggregated materials to HEK cells expressing the A53T -syn variant fused to GFP was found to catalyze -syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.

biochemistry↗

bioRxiv2020

Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aβ42 peptide

Oligomeric aggregates populated during the aggregation of the A{beta}42 peptide have been identified as potent cytotoxins linked to Alzheimers disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach combining theory, experiment, and simulation, we reveal in molecular detail the mechanisms of A{beta}42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we find that most A{beta}42 oligomers dissociate to their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar species. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

biophysics↗

bioRxiv2019

Kinetic fingerprint of antibody therapies predicts outcomes of Alzheimer clinical trials

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-{beta} peptide (A{beta}) is a causative process in Alzheimers disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating A{beta}-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. We bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-A{beta} antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of A{beta}. Our results uncover that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of A{beta}.

biophysics↗