Biology subjects

Kern, A.

Publications and source records attributed to Kern, A..

8 recordsLinked to original sources

bioRxiv2025

Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modelling was used to predict the structure of the SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric 3-4 loop adapted a helically wound conformation to elongate helix 4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix 4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the {beta}1/{beta}2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the {beta}1/{beta}2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved SaMhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus. IMPORTANCES. aureus is a major human pathogen, which can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance towards quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for regulation of uric acid resistance by UrtR family repressors (1). Our results contribute to a better understanding of antimicrobial resistance regulation, which can be exploited for future drug-design to eradicate multidrug-resistant S. aureus.

microbiology↗

bioRxiv2024

The role of the synaptic vesicle protein SV2A in regulating mitochondrial morphology and autophagy.

The synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein of synaptic vesicles. It is involved in key functions of neurons, focused on the regulation of neurotransmitter release. Here we report three novel findings suggesting a completely new role of SV2A. First, we demonstrate that SV2A is localized at the outer mitochondrial membrane (OMM) using confocal and super-resolution microscopy. Second, Inactivation of SV2A in our cell and animal models leads to fragmented mitochondria. In addition, SV2A also affects the basal autophagic flux as well as mitophagy. Third, using proteomics analysis we demonstrate that SV2A interacts with the fission factor DRP1 and the autophagy factor ATG9A. Using AlphaFold3 we provide a first glimpse of the molecular interaction between DRP1 and SV2A. Our findings demonstrate that SV2A is not only a vesicular protein but also a mitochondrial protein in the OMM with defined functions regulating mitochondrial morphology and autophagy.

molecular biology↗

bioRxiv2023

Identification of potential selective autophagy receptors from protein-content profiling of autophagosomes

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts e.g., under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR-docking site (LDS) and Ubiquitin-interacting motif (UIM)-UIM-docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy.

cell biology↗

bioRxiv2022

Accurate Detection of Incomplete Lineage Sorting via Supervised Machine Learning

Gene tree discordance due to incomplete lineage sorting or introgression has been described in numerous genomic datasets. Among distantly related taxa, however, it is difficult to differentiate these biological sources of discordance from discordance due to errors in gene tree reconstruction, even when supervised machine learning techniques are used to infer individual gene trees. Here, rather than applying machine learning to the problem of inferring single tree topologies, we develop a model to infer important properties of a particular internal branch of the species tree via genome-scale summary statistics extracted from individual alignments and inferred gene trees. We show that our model can effectively predict the presence/absence of discordance, estimate the probability of discordance, and infer the correct species tree topology in the presence of multiple, common sources of error. While gene tree topology counts are the most salient predictors of discordance at short time scales, other genomic features become relevant for distantly related species. We validate our approach through simulation, and apply it to data from the deepest splits among metazoans. Our results suggest that the base of Metazoa experienced significant gene tree discordance, implying that discordant traits among current taxa can be explained without invoking homoplasy. In addition, we find support for Porifera as the sister clade to the rest of Metazoa. Overall, these results demonstrate how machine learning can be used to answer important phylogenetic questions, while marginalizing over individual gene tree--and even species tree--topologies.

bioinformatics↗

bioRxiv2022

Genetic Analysis of Wild and Cultivated Populations of Northern Wild Rice (Zizania palustris L.) Reveal New Insights into Gene Flow and Domestication

Northern Wild Rice (NWR; Zizania palustris L.) is an aquatic, annual grass with significant ecological, cultural, and economic importance to the Great Lakes region of North America. In this study, we assembled and genotyped a diverse collection of 839 NWR individuals using genotyping-by-sequencing (GBS) and obtained 5,955 single-nucleotide polymorphisms (SNPs). Our collection consisted of samples from 12 wild NWR populations collected across Minnesota and Western Wisconsin, some of which were collected over two time points; a representative collection of cultivated NWR varieties and breeding populations; and a Zizania aquatica outgroup. Using these data, we characterized the genetic diversity, relatedness, and population structure of this broad collection of NWR genotypes. We found that wild populations of NWR clustered primarily by their geographical location, with some clustering patterns likely influenced by historical ecosystem management. Cultivated populations were genetically distinct from wild populations, suggesting limited gene flow between the semi-domesticated crop and its wild counterparts. The first genome-wide scans of putative selection events in cultivated NWR suggest that the crop is undergoing heavy selection pressure for traits conducive to irrigated paddy conditions. Overall, this study presents a large set of SNP markers for use in NWR genetic studies and provides new insights into the gene flow, history, and complexity of wild and cultivated populations of NWR.

genetics↗

bioRxiv2021

Protein content and lipid profiling of isolated native autophagosomes

Autophagy is a central eukaryotic catabolic pathway responsible for clearance and recycling of an extensive portfolio of cargoes, which are packed in vesicles, called autophagosomes, and are delivered to lysosomes for degradation. Besides basal autophagy, which constantly degrades cellular material, the pathway is highly responsive to several stress conditions. However, the exact protein content and phospholipid composition of autophagosomes under changing autophagy conditions remain elusive so far. Here, we introduce a FACS-based approach for isolation of native unmanipulated autophagosomes and ensure the quality of the preparations. Employing quantitative proteomics and phospholipidomics, we obtained a profound cargo and lipid profile of autophagosomes purified upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Indeed, starvation only mildly affected the content profile, while interference with proteasome activity showed stronger effects and specifically altered autophagosome cargoes. Interestingly, the phospholipid composition of autophagosomes was unaffected by the different treatments. Thus, the novel isolation method enables purification of intact autophagosomes in large quantities and allows protein content and phospholipid profiling without the requirement of exhaustive cellular fractionation or genetic manipulation.

biochemistry↗

bioRxiv2018

RAB18 impacts autophagy via lipid droplet-derived lipid transfer and is rescued by ATG9A

Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability.

cell biology↗

bioRxiv2016

Natural diversity of the malaria vector Anopheles gambiae

The sustainability of malaria control in Africa is threatened by rising levels of insecticide resistance, and new tools to prevent malaria transmission are urgently needed. To gain a better understanding of the mosquito populations that transmit malaria, we sequenced the genomes of 765 wild specimens of Anopheles gambiae and Anopheles coluzzii sampled from 15 locations across Africa. The data reveal high levels of genetic diversity, with over 50 million single nucleotide polymorphisms across the 230 Mbp genome. We observe complex patterns of population structure and marked variations in local population size, some of which may be due at least in part to malaria control interventions. Insecticide resistance genes show strong signatures of recent selection associated with multiple independent mutations spreading over large geographical distances and between species. The genetic variability of natural populations substantially reduces the target space for novel gene-drive strategies for mosquito control. This large dataset provides a foundation for tracking the emergence and spread of insecticide resistance and developing new vector control tools.

genomics↗