Biology subjects

Marcus, K.

Publications and source records attributed to Marcus, K..

13 recordsLinked to original sources

bioRxiv2025

Proteomic Analysis Links Truncated Tau to Lysosome Motility, Autophagy and Endo-lysosomal Dysfunction

Tauopathies are characterized by the progressive accumulation of abnormal tau species, which disrupt the autophagy-lysosomal pathway (ALP), a critical system for degrading intracellular macromolecules and aggregated proteins, causing toxicity and cell death. This study investigates the impact of the N-terminally truncated Tau35 protein overexpression on proteolytic pathways, including effects on autophagy and endo-lysosomal processes. Using a Tau35 mouse model and SH-SY5Y cell lines stably expressing either the Tau35 fragment or full-length tau, we employed western blotting, proteomic analysis of lysosome-enriched brain fractions, proteolysis/endocytosis assays, and live-cell imaging with the lysotracker reporter to assess protein degradation and lysosomal function. Our findings identify early pathological changes in endo-lysosomal processes, including increased endocytosis, proteolytic dysfunction and lysosomal motility abnormalities, associated with Tau35-induced toxicity. This work extends previous research by providing new insights into the mechanisms of Tau35-induced neurotoxicity, offering a foundation for developing targeted therapeutic strategies to address tauopathies.

neuroscience↗

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2024

Schizophrenia risk gene ZNF804A controls ribosome localization and synaptogenesis in developing human neurons

ZNF804A was amongst the first genes robustly associated with schizophrenia based on findings from large-scale genomic studies. Previous research has implicated ZNF804A in the regulation of gene expression and synaptic function, but the role of this gene in neurodevelopment and in schizophrenia pathogenesis remains unclear. To study its function during neurodevelopment, we generated isogenic human induced pluripotent stem cells (hiPSCs) with reduced ZNF804A expression, differentiated them into developing cortical glutamatergic neurons and studied their transcriptomic, synaptic and protein signatures. Mutant neurons showed modest evidence changes in gene expression. However, high-content confocal imaging revealed increased excitatory synapse density in mutant neurons. Cell-compartment specific proteomic analysis further revealed that mutant neurons had higher levels of ribosomal and translational proteins within neurites, and high-content imaging confirmed increased local protein synthesis efficiency. Overall, these results demonstrate that in human developing cortical glutamatergic neurons, ZNF804A regulates excitatory synapse formation potential via increased local protein translation.

neuroscience↗

bioRxiv2023

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy. HighlightsO_LIR349P desminopathy immortalised murine myoblasts as a cellular disease model C_LIO_LIElectrical stimulation improves myofibrillar maturation in desminopathy myotubes C_LIO_LIReduced proton leak in mitochondria of homozygous R349P desmin knock-in myotubes C_LIO_LIReduced ADP/ATP translocase levels in mitochondria of desminopathy myotubes C_LIO_LIEarly signs of secondary mitochondriopathy in desminopathy in cultured myotubes C_LI

cell biology↗

bioRxiv2023

Biological embedding of childhood adversity - a multi-omics perspective on stress regulation

The experience of adversity in childhood can have life-long consequences on health outcomes. In search of mediators of this relationship, alterations of bio-behavioral and cellular regulatory systems came into focus, including those dealing with basic gene regulatory processes. Systems biology oriented approaches have been proposed to gain a more comprehensive understanding of the complex multiple interrelations between and within layers of analysis. We used co-expression based, supervised and unsupervised single and multi-omics system approaches to investigate the influence of childhood adversity on gene expression, protein expression and DNA methylation in CD14+ monocytes of healthy adults before and after exposure to an experimental psychosocial stress protocol. Childhood adversity explained some variance at the single analyte level and within gene and protein co-expression structures. A single-omic, post stress gene expression model differentiated best between participants with a history of childhood adversity and controls in supervised analyses. In unsupervised analyses, a multi-omics based model showed best performance but separated participants based on sex only. Multi-omics analyses are a promising concept but might yield different results based on the specific approach taken and the omic-datasets supplied. Here, stress associated gene-expression pattern were most strongly associated with childhood adversity, and integrating multiple cellular layers did not results in better discriminatory performance. Currently, the capacity and yield of different omics-profiling methods might limit the full potential of integrative approaches.

systems biology↗

bioRxiv2022

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance StatementSARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

microbiology↗

bioRxiv2022

LUBAC assembles a signaling platform at mitochondria for signal amplification and shuttling of NF-ĸB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-B pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-B to the nucleus. TNF induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-B is locally activated and transported to the nucleus by mitochondria, resulting in an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN.

cell biology↗

bioRxiv2022

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

biochemistry↗

bioRxiv2021

GoMi - A new gold standard corpus for miRNA Named Entity Recognition to test dictionary, rule-based and machine-learning approaches.

Biomarkers have been the focus of research for more than 30 years [REF1]. Paone et al. were among the first scientists to use the term biomarker in the course of a comparative study dealing with breast carcinoma [REF2]. In recent years, in addition to proteins and genes, miRNA or micro RNAs, which play an essential role in gene expression, have gained increased interest as valuable biomarkers. As a result, more and more information on miRNA biomarkers can be extracted via text mining approaches from the increasing amount of scientific literature. In the late 1990s the recognition of specific terms in biomedical texts has become a focus of bioinformatic research to automatically extract knowledge out of the increasing number of publications. For this, amongst other methods, machine learning algorithms are applied. However, the recognition (classification) capability of terms by machine learning or rule based algorithms depends on their correct and reproducible training and development. In the case of machine learning-based algorithms the quality of the available training and test data is crucial. The algorithms have to be tested and trained with curated and trustable data sets, the so-called gold or silver standards. Gold standards are text corpora, which are annotated by expertes, whereby silver standards are curated automatically by other algorithms. Training and calibration of neural networks is based on such corpora. In the literature there are some silver standards with approx. 500,000 tokens [REF3]. Also there are already published gold standards for species, genes, proteins or diseases. However, there is no corpus that has been generated specifically for miRNA. To close this gap, we have generated GoMi, a novel and manually curated gold standard corpus for miRNA. GoMi can be directly used to train ML-methods to calibrate or test different algorithms based on the rule-based approach or dictionary-based approach. The GoMi gold standard corpus was created using publicly available PubMed abstracts. GoMi can be downloaded here: https://github.com/mpc-bioinformatics/mirnaGS---GoMi.

bioinformatics↗

bioRxiv2021

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

biophysics↗

bioRxiv2019

Escalating protein supersaturation underlies inclusion formation in muscle proteinopathies

Abundant, aggregation prone or \"supersaturated\" proteins are a feature of neurodegeneration. Whether the principle of supersaturation can similarly explain the widespread aggregation that occurs in non-neuronal protein conformational disorders and underlies pathogenic protein aggregate formation is not established. To test this prediction we analyzed proteomic datasets of biopsies from genetic and acquired protein aggregate myopathy (PAM) patients by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that similar to neurodegeneration, a supersaturated subproteome of aggregate prone proteins is present in skeletal muscle from healthy patients. This subproteome escalates in degree of supersaturation as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion containing fibers, supersaturated proteins within the aggregate subproteome rise in abundance, suggesting they escape normal regulation. We show in the context of a human conformational disorder that the level of supersaturation of a metastable subproteome helps to explain widespread aggregation and correlates with the histopathological state of the tissue.\n\nSignificanceIncreasing evidence implicates the phenomenon of protein supersaturation with the selective vulnerability of specific cells to protein misfolding disorders. Quantitative studies of this phenomenon, however, have only been possible post mortem in the case of neurodegenerative diseases. To overcome this limitation, we study here protein aggregate myopathies (PAMs), for which we were able to carry out systematic single fiber proteomic studies on patient-derived samples. We found not only that proteins associated with PAM inclusions are highly supersaturated in muscle but also that their supersaturation levels increases further in affected fibers. These results provide a clear illustration of how an escalation in supersaturation leads protein inclusions in vulnerable cells.

biophysics↗

bioRxiv2018

Protein inference using PIA workflows and PSI standard file formats

Proteomics using LC-MS/MS has become one of the main methods to analyze the proteins in biological samples in high-throughput. But the existing mass spectrometry instruments are still limited with respect to resolution and measurable mass ranges, which is one of the main reasons why shotgun proteomics is the major approach. Here, proteins are digested, which leads to the identification and quantification of peptides instead. While often neglected, the important step of protein inference needs to be conducted to infer from the identified peptides to the actual proteins in the original sample.\n\nIn this work, we highlight some of the previously published and newly added features of the tool PIA - Protein Inference Algorithms, which helps the user with the protein inference of measured samples. We also highlight the importance of the usage of PSI standard file formats, as PIA is the only current software supporting all available standards used for spectrum identification and protein inference. Additionally, we briefly describe the benefits of working with workflow environments for proteomics analyses and show the new features of the PIA nodes for the KNIME Analytics Platform. Finally, we benchmark PIA against a recently published dataset for isoform detection.\n\nPIA is open source and available for download on GitHub (https://github.com/mpc-bioinformatics/pia) or directly via the community extensions inside the KNIME analytics platform.

bioinformatics↗

bioRxiv2018

Chemical signatures of honey bee group membership develop via a socially-modulated innate process

Large social insect colonies exhibit a remarkable ability for recognizing group members via colony-specific cuticular hydrocarbon (CHC) pheromonal signatures. Previous work suggested that in some ant species colony-specific signatures are generated through a \"gestalt\" mechanism via the passive transfer and homogenization of CHCs across all individual members of the colony. In contrast, we demonstrate that nestmate recognition cues of worker honey bees (Apis mellifera) mature in foragers via a sequence of stereotypic age-dependent quantitative and qualitative chemical transitions, which are driven by intrinsic biosynthetic pathways. Therefore, in contrast to predictions of the \"Gestalt\" model, nestmate recognition cues in honey bee colonies do not represent a passive \"average\" signature that is carried and recognized by all colony members. Instead, specific colony members develop the relevant cues via an innately-determined developmental program that can be modulated by colony-specific social environmental factors.

animal behavior and cognition↗