Biology subjects

Kaplan, E.

Publications and source records attributed to Kaplan, E..

7 recordsLinked to original sources

bioRxiv2026

The multidrug transporter Pdr5 from Saccharomyces cerevisiae is an ergosterol-modulated efflux pump

The pleiotropic drug resistance ABC transporter Pdr5 and its homologs play a critical role in multidrug resistance of numerous fungi, including pathogens. Previous studies demonstrated that alterations in the composition of the plasma membrane in which Pdr5 is embedded, specifically the ergosterol content, affected transport activity. Here we report the cryo-EM map of Saccharomyces cerevisiae Pdr5 in complex with ATP/ADP at 2.2 [A] resolution, revealing densities on the surface of the transmembrane domains as well as inside the substrate-binding site, that were assigned as ergosterol, identified and quantified by GC-MS, and the sphingolipid M(IP)2C, identified via native MS. The ergosterol molecules are bound to the protein in well-defined grooves and unexpected locations. We found that the activity of Pdr5 follows a Gaussian-like distribution, increasing with the ergosterol-concentration to reach a maximum before declining at higher concentrations, but this modulation was not observed in the presence of cholesterol, the sterol analogue found in mammalian cells. Mutational analysis corroborates the role of a single ergosterol-binding site in the modulation of Pdr5 activity. Our structural analysis identifies a protein-lipid network that could serve as a center for lipid engagement linking the surrounding lipid environment to the substrate-binding region of Pdr5. These findings advance our understanding of Pdr5 function by demonstrating that the lipid environment is closely linked to Pdr5 structure and activity.

biochemistry↗

bioRxiv2026

Structures of LolB bound to LolA or lipoprotein resolve the final steps of bacterial lipoprotein trafficking

In Gram-negative bacteria, lipoproteins are structural elements of the outer membrane and essential components of machineries responsible for its construction and maintenance. The Lol system, responsible for the trafficking of lipoproteins from the site of maturation on the inner membrane to the outer membrane, is therefore crucial to the function of the cell envelope and a key target of efforts to find novel antimicrobials. In the final steps of this process, the outer membrane receptor, LolB accepts triacylated lipoproteins from the periplasmic chaperone LolA before inserting them into the outer membrane. Here we present a structure of LolB in complex with LolA, validated by in vivo and in vitro assays, highlighting how positively charged residues on the convex face of the LolB {beta}-barrel underpin complex formation. A protruding loop of LolB, essential for function, inserts into the LolA cavity in position to initiate the displacement of substrate lipoprotein from LolA to enable transfer to LolB. Structural resolution of a lipoprotein-bound LolB complex in combination with biophysical assays shows how a molecular latch releases the lid of the cavity to accommodate the lipoprotein acyl chains. Modelling of these structures onto computationally predicted orientations for LolB on the outer membrane provides a rationale for LolA release and lipoprotein triacyl group membrane insertion. Taken altogether, our data elucidate atomic resolution of two key intermediates and provide a greater understanding of the terminal steps of lipoprotein trafficking events at the bacterial outer membrane.

microbiology↗

bioRxiv2025

A lipoprotein partner for the Escherichia coli outer membrane protein TolC

The outer-membrane protein TolC from Escherichia coli belongs to an extensive superfamily whose members are found throughout the didermal, Gram-negative bacterial lineages. The protein serves as an activated exit duct in multi-drug efflux pumps and protein secretion machinery. Many TolC homologs bear a lipid modification on the N-terminus that embeds into the inner leaflet of the outer membrane and appears to have been a conserved feature; however, the moiety is absent entirely in the E. coli TolC. We have discovered that the E. coli lipoprotein YbjP interacts extensively with the periplasmic surface of TolC and its N-terminal lipid moiety is embedded in the membrane, mimicking the intramolecular and modification-membrane interactions seen in TolC homologs. Here, we present cryo-EM structures of the MacA-MacB-TolC and AcrA-AcrB-TolC tripartite pumps complexed to YbjP. Although the association occurs spontaneously both in vitro and in vivo, the YbjP-TolC interaction is not required for efflux activity under standard laboratory conditions. YbjP may contribute stabilising the orientation and distribution of TolC in the outer membrane as well as the expression of transporters for tryptophan and cyclic peptide toxins.

molecular biology↗

bioRxiv2024

Neolithic introgression of IL23R-related protection against chronic inflammatory bowel diseases in modern Europeans

BackgroundThe hypomorphic variant rs11209026-A in the IL23R gene provides significant protection against immune-related diseases in Europeans, notably inflammatory bowel disease (IBD). Today, the A-allele occurs with an average frequency of 5% in Europe. MethodsThis study comprised 251 ancient genomes from Europe spanning over 14,000 years. In these samples, the investigation focused on admixture informed analyses and selection scans of rs11209026-A and its haplotypes. Findingsrs11209026-A was found at high frequencies in Anatolian Farmers (AF, 18%) where it was likely under weak positive selection. AF later introduced the allele into the ancient European gene-pool. Subsequent admixture caused its frequency to decrease and formed the current southwest-to-northeast allele frequency cline in Europe. The geographic distribution of rs11209026-A may influence the gradient in IBD incidence rates that are highest in northern and eastern Europe. InterpretationGiven the dramatic changes from hunting and gathering to agriculture during the Neolithic, AF might have been exposed to selective pressures from a pro-inflammatory lifestyle and diet. Therefore, the protective A-allele may have increased survival by reducing intestinal inflammation and microbiome dysbiosis. The adaptively evolved function of the variant likely contributes to the high efficacy and low side-effects of modern IL-23 neutralization therapies for chronic inflammatory diseases. This study highlights how evolutionary informed research can provide promising targets for new therapeutic strategies. FundingDeutsche Forschungsgemeinschaft (DFG German Research Foundation) under Germanys Excellence Strategy - EXC 2167 390884018 and EXC 2150 390870439.

evolutionary biology↗

bioRxiv2024

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia

Alzheimers disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-{beta} and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher ([~]two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

genomics↗

bioRxiv2019

Highly clustered de novo frameshift variants in the neuronal splicing factor NOVA2 result in a specific abnormal C terminal part and cause a severe form of intellectual disability with autistic features.

The Neuro-Oncological Ventral Antigen 2 NOVA2 protein is a major factor regulating neuron specific alternative splicing, previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in the NOVA2 gene affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait and abnormal brain MRI. The six variants lead to the same reading frame, adding a common 133 aa long proline rich C-terminus part instead of the last KH RNA binding domain. We detected forty-one genes differentially spliced after NOVA2 inactivation in human neural cells. The mutant NOVA2 protein shows decreased ability to bind a target RNA, to regulate specific splicing events and to rescue the phenotype of altered retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss of function, although a specific contribution of the novel C terminal extension cannot be excluded on the basis of the genetic findings.

genetics↗

bioRxiv2017

Systems Therapeutics for Axonal Regeneration in the Central Nervous System

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axon using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Morphology experiments show that the four-drug combination promotes axonal regrowth to the optic chiasm and beyond. Physiologically, drug treatment restored pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. We conclude that spatially targeted drug treatment can promote robust axonal regeneration and can restore limited functional recovery.

neuroscience↗