Biology subjects

Holzer, M.

Publications and source records attributed to Holzer, M..

6 recordsLinked to original sources

bioRxiv2025

Customized Protein Nanoreactors for Affibody-Directed Activation of 5-Fluorcytosin in HER2-Positive Cells

The development of targeted therapies for HER2-positive cancers remains critical due to resistance and toxicity challenges in current treatments. Here, we present the rational engineering of protein-based encapsulin nanocompartments for selective catalytic prodrug activation. Encapsulins provide precise cargo loading, exceptional stability, and versatile engineerability, making them ideal nanoreactors for therapeutic applications. Our encapsulin constructs encapsulate tandem cytosine deaminase enzymes and display HER2-specific affibodies on their exterior, enabling precise cellular targeting. These engineered nanoreactors catalyze the efficient conversion of the prodrug 5-fluorocytosine (5-FC) into the cytotoxic agent 5-fluorouracil (5-FU), yielding an 83% reduction in viability of HER2-overexpressing SKOV3 cells. Structural characterization using native gel electrophoresis confirms stable assembly with functional affibody presentation. This enzyme-prodrug approach showcases how supramolecular protein architectures can serve as customizable platforms for affibody-directed, enzyme-mediated therapy, offering a promising strategy to enhance therapeutic specificity and minimize systemic side effects in HER2-positive cancer treatment. Significance StatementThis work establishes encapsulins as a new class of programmable therapeutic nanoreactors by integrating selective affibody-mediated targeting with enzyme-prodrug catalysis in HER2-positive cancer cells. Unlike virus-like particles, which often lack precise cargo loading and structural robustness, encapsulins enable dual engineering of both interior and exterior domains for stable, multifunctional assemblies. Compared to antibody-drug conjugates, these nanoreactors achieve amplified drug generation through localized prodrug activation, overcoming payload limitations and reducing systemic toxicity. This platform introduces a versatile supramolecular strategy for targeted cancer therapy that exceeds current delivery technologies.

cancer biology↗

bioRxiv2023

Pangenome calculation beyond the species level using RIBAP: A comprehensive bacterial core genome annotation pipeline based on Roary and pairwise ILPs

Pangenome analysis is a computational method for identifying genes that are present or absent from a group of genomes, which helps to understand evolutionary relationships and to identify essential genes. While current state-of-the-art approaches for calculating pangenomes comprise various software tools and algorithms, these methods can have limitations such as low sensitivity, specificity, and poor performance on specific genome compositions. A common task is the identification of core genes, i.e., genes that are present in (almost) all input genomes. However, especially for species with high sequence diversity, e.g., higher taxonomic orders like genera or families, identifying core genes is challenging for current methods. We developed RIBAP (Roary ILP Bacterial core Annotation Pipeline) to specifically address these limitations. RIBAP utilizes an integer linear programming (ILP) approach that refines the gene clusters initially predicted by the pangenome pipeline Roary. Our approach performs pairwise all-versus-all sequence similarity searches on all annotated genes for the input genomes and translates the results into an ILP formulation. With the help of these ILPs, RIBAP has successfully handled the complexity and diversity of Chlamydia, Klebsiella, Brucella, and Enterococcus genomes, even when genomes of different species are part of the analysis. We compared the results of RIBAP with other established and recent pangenome tools (Roary, Panaroo, PPanGGOLiN) and showed that RIBAP identifies all-encompassing core gene sets, especially at the genus level. RIBAP is freely available as a Nextflow pipeline under the GPL3 license: https://github.com/hoelzer-lab/ribap.

bioinformatics↗

bioRxiv2022

Extensive genomic divergence among 61 strains of Chlamydia psittaci

Chlamydia (C.) psittaci, the causative agent of avian chlamydiosis and human psittacosis, is a genetically heterogeneous species. Its broad host range includes parrots and many other birds, but occasionally also humans (via zoonotic transmission), ruminants, horses, swine and rodents. To assess whether there are genetic markers associated with host tropism we comparatively analyzed whole-genome sequences of 61 C. psittaci strains. Initially, poorly assembled genomes in public databases were subjected to clean-up, reassembly and polishing. Multiple sequence alignment of the genome sequences revealed four major clades within this species. Clade 1 represents the most recent lineage comprising 40/61 strains and contains 9/10 of the psittacine strains, including type strain 6BC, and 10/13 of human isolates. Clades 2-4 carry strains from different non-psittacine hosts. We found that clade membership correlates with classification schemes based on SNP types, ompA genotypes, multilocus sequence types as well as plasticity zone (PZ) structure and host preference. Genome analysis also revealed that i) sequence variation in the major outer membrane porin OmpA can result in 3D structural changes of immunogenic domains, ii) past host change of Clade 3 and 4 strains could be associated with loss of MAC/perforin in the PZ, rather than the large cytotoxin, iii) the distinct phylogeny of atypical strains (Clades 3 and 4) is also reflected in their repertoire of inclusion proteins (Inc family) and polymorphic membrane proteins (Pmps). Altogether, our study identified a number of genomic features that can be correlated with the phylogeny and host preference of C. psittaci strains.

genomics↗

bioRxiv2022

Tau and α-synuclein shape microtubule organization and microtubule-dependent transport in neuronal dendrites

Tau and -synuclein are major players in neurodegenerative diseases, but their physiological role, particularly in dendrites, is poorly understood. Here we show that, surprisingly, lack of tau protein induces the development of a more elaborate dendritic arbor of hippocampal pyramidal cells in organotypic tissue. Using high-speed volumetric lattice light-sheet microscopy and single particle tracking, we found a more directional KIF1A-mediated transport in dendrites of Tau KO cells. Increased transport processivity correlated with longer and straighter dendritic microtubules as revealed by three-dimensional super-resolution microscopy of cultured hippocampal neurons. Unbiased mass spectrometric analysis of tissue showed highly increased expression of -synuclein in Tau KO hippocampi. Overexpression of -synuclein mimicked the transport characteristics observed in Tau KO cells. Our data indicate that tau and -synuclein shape microtubule-dependent transport in neuronal dendrites, thereby promoting dendritic arborization during maturation. Furthermore, the data demonstrate that transport efficiency and length and straightness of microtubules are correlated.

neuroscience↗

bioRxiv2020

FcγR responses to soluble immune complexes of varying size: A scalable cell-based reporter system

Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings. One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings.

immunology↗

bioRxiv2019

Preparation and characterization of phospholipid stabilized nanoemulsions in small-scale

Phospholipids have been used to prepare liposomes. The use of phospholipids to stabilize nanoemulsions may cause spontaneous formation of liposomes. The main objective of this study is to develop a method to prepare phospholipid stabilized nanoemulsions in small scale (< 1 mL) and to minimize the formation of liposomes.\n\nA combination of hand extrusion and detergent removal methods was used in this study. Extrusion through polycarbonate membranes was performed in two steps, firstly using membranes of 400 nm followed by 200 nm membranes as the second step. Sodium cholate was used as a detergent to solubilize the formed liposomes which was later removed via dialysis. Nanoemulsions were characterized by measuring their particle size, polydispersity index and zeta-potential using Photon Correlation Spectroscopy and Cryo-TEM pictures. The stability of nanoemulsion stored under refrigeration was also studied.\n\nFifty-one extrusion cycles through polycarbonate membrane of 400 nm pore size followed by one-hundred fifty-three cycles through polycarbonate membrane of 200 nm produced nanoemulsions having particle size below 200 nm (diameter). The nanoemulsions were found to be homogenous as depicted by polydispersity index (PDI) value below 0.1. Similarly, the zeta-potential was measured to be above -30 mV which is sufficient to keep nanoemulsions stable for as long as 7 months when stored under refrigeration. The Cryo-TEM pictures revealed 30 mM to be an optimum concentration of sodium cholate to prepare homogenous nanoemulsions with negligible proportion of liposomes.\n\nIt was concluded that this method could be established as a small scale method of preparing nanoemulsions which will not only reduce the cost of preparation but also the disposal cost of toxic chemicals used for functionalizing nanoemulsions for scientific research.

biophysics↗