Biology subjects

Bothe, S.

Publications and source records attributed to Bothe, S..

5 recordsLinked to original sources

bioRxiv2025

Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

A hallmark of Alzheimers disease (AD), the most common form of dementia, is the progressive accumulation of amyloid-beta (A{beta}) peptides across distinct brain regions. Anti-A{beta} antibodies (A{beta}-Abs) targeting specific A{beta} variants are essential tools for AD research, diagnostics, and therapy. The monoclonal antibodies Aducanumab, Lecanemab, and Donanemab have recently been approved as the first disease-modifying treatments for early AD, highlighting the clinical importance of their exact binding profiles. In this study, we systematically characterized the binding and modification requirements of 20 A{beta}-Abs, including biosimilars of Aducanumab, Lecanemab, and Donanemab, across monomeric, oligomeric, and aggregated A{beta} forms. Array-based analysis of 20,000 modified A{beta} peptides defined binding epitopes at single-residue resolution and revealed the impact of sequence variation, including familial AD mutations, as well as diverse post-translational modifications (PTMs). Notably, genetic variants such as H6R impaired binding of therapeutic A{beta}-Abs like Aducanumab. Donanemab showed strong preference for pyroglutamate-modified A{beta}pE3-10, while Lecanemab and Aducanumab exhibited aggregation- and sequence-context-dependent binding requirements. Comparison of peptide binding profiles with binding of full-length and aggregated A{beta} via immunoprecipitation-mass spectrometry, capillary immunoassays, Western blotting, and immunohistochemistry on AD brain tissue revealed distinct aggregation-dependent binding behaviours. The valency- and context-dependence of Aducanumab binding, together with its preference for Ser8-phosphorylated A{beta}, supports a dimerization-mediated binding mechanism. For Lecanemab, our data suggest that additional structural contributions beyond the minimal N-terminal epitope are required for binding to aggregated A{beta}, which remain to be fully resolved. Together, this work provides the most comprehensive dataset to date on aggregation-dependent sequence and modification selectivity of A{beta}-Abs. By integrating mutational, PTM, and aggregation contexts in a unified experimental framework, we establish a resource that enables rational selection of antibodies for research and diagnostic applications, and offers mechanistic insights that may inform the design and optimization of future therapeutic antibodies in AD.

biochemistry↗

bioRxiv2024

A genome-wide RNA interference screening reveals protectiveness of SNX5 knockdown in a Parkinson`s disease cell model

BackgroundAlpha-synuclein is a major player in the pathophysiology of a group of diseases called synucleinopathies, which include Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. To date, there is no disease-modifying therapy available for these synucleinopathies. Furthermore, the intracellular mechanisms by which alpha-synuclein confers toxicity are not yet fully understood. Therefore, it is of utmost importance to investigate the pathophysiology of alpha-synuclein-induced toxicity in order to identify novel molecular targets for the development of disease-modifying therapies. MethodsIn the present study, we performed the first genome-wide siRNA modifier screening in a human postmitotic neuronal cell model using alpha-synuclein-induced toxicity as read-out. In a multi-step approach, we identified several genes, whose knockdown protected from alpha-synuclein-induced toxicity. The main hit was further validated by different methods, including immunofluorescence microscopy, qPCR, and Western blot. ResultsThe highest protection was achieved by knockdown of SNX5, which encodes the SNX5 protein, a component of the retromer complex. We confirmed the protective efficacy of SNX5 knockdown with an independent siRNA system. SNX5 protein is part of SNX-BAR heterodimers, which are part of the retromer complex. We found that extracellular and overexpressed intracellular alpha-synuclein led to fragmentation of the trans-Golgi network, which was prevented by SNX5 knockdown by confining alpha-synuclein in early endosomes. ConclusionIn summary, our data suggest that SNX5 plays an important role in trafficking and toxicity of alpha-synuclein. Therefore, SNX5 appears to be a possible target for therapeutic interventions in synucleinopathies.

neuroscience↗

bioRxiv2023

7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover, visualize and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

biochemistry↗

bioRxiv2022

Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97

Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. Here, we describe a biolayer interferometry-based fragment screen targeting the AAA+ ATPase p97, an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors target its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. We demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.

biochemistry↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗