Biology subjects

Schneider, M. M.

Publications and source records attributed to Schneider, M. M..

4 recordsLinked to original sources

bioRxiv2020

In vitro measurements of protein-protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2-spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the binding energies of the binary interactions between the ACE2 receptor and the spike protein, and the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential areas of application ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.

biochemistry↗

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

Microfluidic Antibody Affinity Profiling for In-Solution Characterisation of Alloantibody - HLA Interactions in Human Serum

The detection and characterisation of antibodies in human blood is a key for clinical diagnostics and risk assessment for autoimmunity, infectious diseases and transplanta-tion. Antibody titre derived from immunoassays is a commonly used measure for anti-body response, but this metric does not resolve readily the two fundamental properties of antibodies in solution, namely their affinity and concentration. This difficulty originates from the fact that the fundamental parameters describing the binding interaction, affinity and ligand concentration, are convoluted into the titre measurement; moreover, the difficulty of controlling the surface concentration and activity of the immobilised ligand can make it challenging to distinguish between avidity and affinity. To address these challenges, we developed microfluidic antibody affinity profiling, an assay which allows the simultaneous determination of both affinity and antibody concentration, directly in solution, without surface immobilisation or antibody purification. We demonstrate these measurements in the context of alloantibody characterisation in organ transplantation, using complex patient sera, and quantify the concentration and affinity of alloantibodies against donor Human Leukocyte Antigens (HLA), an extensively used clinical biomarker to access the risk of allograft rejection. These results outline a path towards detection and in depth profiling of antibody response in patient sera.

biophysics↗

bioRxiv2020

Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses

The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and A{beta}42 peptide in Alzheimers disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of A{beta}42 amyloids. Kinetic analysis further corroborates that the surfaces available for the A{beta}42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to A{beta}42 fibrils is validated by a microfuidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.

biophysics↗