Biology subjects

Hansen, T.

Publications and source records attributed to Hansen, T..

At least 19 recordsLinked to original sources

bioRxiv2026

multiomeR: a pipeline for reproducible analysis of large-scale single-nucleus Epi Multiome datasets

The 10x Genomics Epi Multiome assay measures gene expression and chromatin accessibility in the same nuclei, and studies increasingly profile many samples. Yet analysis after Cell Ranger ARC is usually assembled by hand from separate tools, which makes it slow to revise, hard to reproduce and memory-intensive for large studies. multiomeR addresses these problems with a configurable R pipeline for quality control, processing and integration of both data types, and cell-type annotation across many samples. Plain-text configuration files record every analytical choice, each step is tracked so that a change reruns only what it affects, and data stay on disk rather than in memory. Optional modules compare groups of donors, link accessible regions to the genes they may regulate, and test which cell types are enriched for genetic associations with a trait. In a benchmark on 31 public samples (375,757 nuclei), multiomeR had a shorter estimated run time than a Seurat v5.6/Signac v2 workflow (2.2 versus 5.6 h), because it runs independent steps in parallel. It also used fewer memory gigabyte-hours (87 versus 114 GB-hours), with similar total CPU time. multiomeR thus makes large multi-sample multiome studies practical to analyze, revise and extend.

bioinformatics↗

bioRxiv2026

Cryptic Insect Microbiome Compositions Unveiled with Full-Length 16S Sequencing

Insects have interconnected relationships with gut microorganisms which can have impacts on behavior and survival. Understanding foundational ecological principles of the insect-associated microorganisms is important to understand how insects utilize microbes to cope with stress and different environments. Common microbiome surveys of insect microbiomes utilize 16S rRNA sequencing of metagenomic DNA. For amplicon-based surveys of insect microbiome, fragment length and 16S rRNA subunit choice may have unintended biases, and some primer combinations may under-represent genus or species richness. Contemporary solutions target sequencing of the entire 16S rRNA fragment. Here, we illustrate the benefit of full-length 16S rRNA sequencing in improving sample resolution compared to V4 which provides new insight into the gut microbiome community composition of an invasive insect. We evaluated the gut microbiome of mass-reared medfly males (Mediterranean fruit fly, Ceratitis capitata) that were collected across a nine-month sampling period. Full-length 16S rRNA PCR products of samples were prepared into a Kinnex 16S rRNA library, sequenced on a PacBio Revio system, and the resulting HiFi amplicon data was processed into amplicon sequence variants (ASVs). Our findings reveal substantial differences in bacterial compositions across different medfly cohorts when sampling the full-length amplicons which were not detectible when only considering the V4 regions. Strain-level gut microbiome variation were supported with genomes assembled from shotgun metagenomic sequencing. Our findings support that long-read sequencing of full-length 16S rRNA amplicon uncover ecologically important interactions between host and gut microbiomes and serve as a bridge between short-fragment sequencing and shotgun metagenomics.

microbiology↗

bioRxiv2025

Bioinformatic process for the identification and characterization of bacterial repeat-in-toxin adhesins

Gram-negative bacteria attach to host surfaces using ligand-binding domains (LBDs) at the distal tips of fibrillar RTX adhesins. Blocking the initial binding interaction(s) can potentially prevent colonization and subsequent biofilm formation and infection. To this end, adhesins must be identified, and it is also essential to determine the dominant adhesin type for those species that have more than one. RTX adhesins are frequently the largest proteins within each species (ranging from 1500 to 15,000 aa) and are often misannotated as incomplete/pseudogene products because their highly repetitive nature confounds genome assemblies from short-read technologies. Our bioinformatic process collates predicted proteins from long-read assemblies, which are then clustered based on the similarity of their C-terminal regions where the LBDs are typically located. RTX adhesins are identified by their length and domain structure and are modelled using AlphaFold3. An exhaustive search of multiple strains from seven species revealed a total of 35 different RTX adhesins that map to 16 different loci, with differing arrangements of LBDs that include putative carbohydrate-binding modules and von Willebrand Factor A-like domains. Notably, similar adhesins are sometimes found in multiple species, either by descent or through DNA uptake, and three species have an RTX adhesin of uncertain function because it lacks an obvious LBD. ImportanceMany bacteria initiate infection by reaching out with large, complex proteins called adhesins to attach themselves to a host cell. The DNA sequences of adhesins are difficult to read, due to their length and repetitiveness. This study leverages recent technological advances like "long-read sequencing" and structural modelling to identify and characterize the adhesins in seven species of harmful bacteria: Acinetobacter baumannii, Aeromonas hydrophila, Aeromonas salmonicida, Bordetella parapertussis, Legionella pneumophila, Vibrio parahaemolyticus, and Vibrio vulnificus. We identified thirty-five unique versions of adhesins and demonstrated their mix-and-match architecture. This research provides a foundation for strategies to block bacteria from binding surfaces, offering a vital alternative treatment as antibiotic resistance continues to rise.

biochemistry↗

bioRxiv2025

Genomic and Immunogenomic Profiling of Extramedullary Acute Myeloid Leukemia Reveals Actionable Clonal Branching and Frequent Immune Editing

Extramedullary acute myeloid leukemia (eAML) is a rare form of myeloid neoplasm characterized by leukemic infiltration outside the bone marrow (BM). Despite its prognostic significance, eAML is often underdiagnosed and poorly characterized at molecular level. We performed a comprehensive genomic and immunogenomic profiling on paired BM and extramedullary specimens from 26 eAML patients, alongside over 400 AML cases without extramedullary involvement and 97 healthy controls. Clonal branching from BM was observed in 38.5% of extramedullary sites, frequently involving actionable mutations in FLT3, IDH2 and NPM1 genes. Both compartments were enriched in RAS pathway mutations and class II HLA losses, suggesting active immunoediting mechanisms driving eAML development. Strikingly all relapsed cases acquired FLT3 aberrations, highlighting therapeutic opportunities. These findings underpin the need for improved detection and routine genomic profiling, including targeted sequencing of suspected extramedullary lesions.

genomics↗

bioRxiv2024

Sucrase isomaltase dysfunction influences dietary sucrose intake and preference

ObjectiveTo characterise the role of Sucrase-isomaltase (SI) in regulating dietary behaviours, such as sweet preference and food liking in Si knockout (Sis-KO) mice and in population-based cohorts from Greenland and the UK. DesignWe profiled the appetitive and post-ingestive response to dietary carbohydrates in SI knockout (Sis-KO) mice. Alongside this, we conducted detailed dietary analysis of 45 foods in two Greenlandic population-based cohorts (IHIT, n=2778 and 68 foods, and B2018, n=2203 and 45 foods) with the presence of a common (allele frequency = 14.3%) SI Loss of function (LoF) variant, c.273-274delAG. Finally, we explored the association between SI hypomorphic variants, liking of 140 foods, and sucrose content using data from 134,766 UKBB participants with exome sequencing and questionnaire data available. ResultsSucrose naive Sis-KO mice had a significantly reduced intake of dietary sucrose, and preference for 10% liquid sucrose, in two-bottle preference studies. Mechanistically, oral administration of the short-chain fatty acid acetate reduced sucrose-preference in wild-type mice. In Greenlandic LoF homozygous carriers we show that the previously reported reduction in sugar intake may primarily be explained by a lower intake of cake and pastries, and of candy and chocolate and that added sugar is the main factor explaining these associations. In the UKBB, a negative association with "cake icing", the food with the highest sucrose content per 100g, was detected in SI hypomorphic carriers, as well as in sensitivity analyses conducted only including carriers of known CSID LoF variants. Further, a negative linear relationship was also observed between the effect estimates of hypomorphic SI variants on food liking and the estimated sucrose content per 100g of 88 sucrose-containing foods, indicating that food dislike in SI carriers correlates with the amount of sucrose in food. ConclusionCollectively, we demonstrated that genetic variation in the SI gene is associated with significant changes in sucrose preference, characterised by a rapid avoidance of dietary sucrose in Sis-KO mice, as well as lower consumption and increased disliking of sucrose rich foods in Greenlanders and Europeans, respectively. This work demonstrates that genetic variation in the SI gene may impact physiology beyond the gastrointestinal tract and suggest the possibility to target SI to reduce the preference, and intake, of dietary sucrose with implications for digestive and metabolic health.

genetics↗

bioRxiv2024

Fecal microbial load is a major determinant of gut microbiome variation and a confounder for disease associations

The microbiota in individual habitats differ both in relative composition and absolute abundance. While sequencing approaches determine only the relative abundances of taxa and genes, experimental techniques for absolute abundance determination are rarely applied to large-scale microbiome studies. Here, we developed a machine learning approach to predict fecal microbial loads (microbial cells per gram) solely from relative abundance data. Applied to large-scale datasets (n = 34,539), we demonstrate that microbial load is the major determinant of gut microbiome variation and associated with numerous host factors. We found that for several diseases, the altered microbial load, not the disease itself, was the main driver of the gut microbiome changes. Adjusting for this effect substantially reduced the significance of more than half of the disease-associated species. Our analysis reveals that the fecal microbial load is a major confounder in microbiome studies, highlighting its importance for understanding microbiome variation in health and disease.

microbiology↗

bioRxiv2023

Highly conserved, positively charged tandem repeats mediate multimerization of ice nucleation proteins

In nature, frost can form at a few degrees below 0 {degrees}C. However, this process requires the assembly of tens of thousands of ice-like water molecules that align together to initiate freezing at these relatively high temperatures. Water ordering on this scale is mediated by the ice nucleation proteins of common environmental bacteria like Pseudomonas syringae and P. borealis. However, individually, these 100-kDa proteins are too small to organize enough water molecules for frost formation, and it is not known how giant, megadalton-sized multimers, which are crucial for ice nucleation at high sub-zero temperatures, form. The ability of multimers to self-assemble was suggested when the transfer of an ice nucleation protein gene into Escherichia coli led to efficient ice nucleation. Here we demonstrate that a positively-charged sub-domain at the C-terminal end of the central beta-solenoid of the ice nucleation protein is crucial for multimerization. Truncation, relocation, or change of the charge of this subdomain caused a catastrophic loss of ice nucleation ability. Cryo-electron tomography of the recombinant E. coli showed that the ice nucleation protein multimers form fibres that are [~] 5 nm across and up to 200 nm long. A model of these fibres as an overlapping series of antiparallel dimers can account for all their known properties and suggests a route to making cell-free ice nucleators for biotechnological applications.

biochemistry↗

bioRxiv2023

Metabolomics reveals nucleoside analogs for regulating mucosal-associated invariant T cell responses

Although mucosal-associated invariant T (MAIT) cells recognize riboflavin-like metabolites from Gram-negative bacteria, MAIT cell stimulation by broad bacterial families and mammalian cells suggests the existence of novel ligands from different biological sources. Here we established a comparative platform of functional metabolomics and used Mycobacterium tuberculosis as a model to characterize novel metabolites for MAIT cell activation. We extracted and fractionated small metabolites of M. tuberculosis using high-performance liquid chromatography, showing a different MAIT cell stimulation pattern of M. tuberculosis metabolite fractions in comparison with Escherichia coli fractions. Mass profiling predicted multiple nucleoside analogs enriched in a biologically active fraction of M. tuberculosis. Whereas the synthetic forms of these predicted M. tuberculosis nucleosides were unavailable, structural-based autodocking of analogous nucleosides conserved in mammals supported potential binding with MR1 protein. Indeed, functional assays of these conserved nucleosides demonstrated guanosine as a stimulator and deoxyformyluridine as an inhibitor of MAIT cell activation. Identification of bioactive nucleoside metabolites broadly conserved in bacterial and mammalian systems will facilitate an understanding of the regulatory roles of MAIT cells in infectious and inflammatory conditions.

immunology↗

bioRxiv2022

A multiplexed assay of human glucokinase reveals thousands of potential disease variants with both decreased and increased activity

Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia (HH) or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. Here we exploited a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlated with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants were concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shifted the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.

genetics↗

bioRxiv2021

High-throughput UHPLC-MS to screen metabolites in feces for gut metabolic health

(1) BackgroundFeces are the product of our diets and have been linked to diseases of the gut, including Chrons disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection. (2) MethodsAs sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n= 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism. (3) ResultsMS analyses of the prepared fecal samples have shown reproducibility and coverage of n=28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD, LOQ and range of linearity. The average concen-trations for 135 healthy participants are reported here for clinical applications. (4) Conclusionsour high-throughput method provides an efficient tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.

biochemistry↗

bioRxiv2021

Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1 Rev-Rev interaction

Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing NLUC-Rev or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z) for the cell-free system was ~0.85 when tested in 96-well format, and anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.

molecular biology↗

bioRxiv2021

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by promoting RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 loading to sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.

cell biology↗

bioRxiv2020

SorCS1 binds the insulin receptor to enhance insulin sensitivity

Type 2 diabetes has reached endemic proportions and is a substantial burden for the affected patients and the society. Along with lifestyle factors, a number of genetic loci predisposing to type 2 diabetes have been identified, including SORCS1 that encodes the transmembrane receptor SorCS1. The ectodomain of SorCS1 (sol-SorCS1) is shed from plasma membranes but the biological function of this fragment is unknown. Here we show that sol-SorCS1 acts as a high-affinity binding partner for the insulin receptor to stabilize the receptor and increase insulin affinity, protein kinase B activation, and glucose uptake in myocytes. Sol-SorCS1 is liberated from adipocytes, and in diabetic patients the plasma concentration positively correlates with body mass index, but inversely with plasma glucose. In mouse models of insulin resistance, exogenous sol-SorCS1 restored insulin sensitivity. We conclude that sol-SorCS1 increases peripheral insulin sensitivity and propose sol-SorCS1 as a novel insulin sensitizing adipokine and potential antidiabetic agent.

physiology↗

bioRxiv2020

A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease

Existing tests for detecting liver fibrosis, inflammation and steatosis, three stages of liver disease that are still reversible are severely hampered by limited accuracy or invasive nature. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to identify biomarkers in a cross-sectional alcohol-related liver disease cohort of nearly 600 individuals. Metabolic functions were downregulated whereas fibrosis-associated signaling and novel immune responses were upregulated, but only half of tissue proteome changes were transmitted to the circulation. Machine learning models based on our biomarker panels outperformed existing tests, laying the foundation for a generic proteomic liver health assessment.

biochemistry↗

bioRxiv2020

Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes in Saccharomyces cerevisiae

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may help S. cerevisiae cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPTSperoxisomal targeting signal;LC-MSliquid chromatography-mass spectrometry;TEVtobacco etch virus;TPATEV protease cleavage site-Protein AView Full Text

cell biology↗

bioRxiv2020

Nutrient sensing pathways regulating adult reproductive diapause in C. elegans

Genetic and environmental manipulations, such as dietary restriction (DR), can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when DR occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a DR model that is associated with lengthened lifespan and reproductive potential (Angelo and Van Gilst 2009). As the metabolic pathways regulating ARD have not yet been explored in depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we find that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. We also discovered that fatty acid {beta}-oxidation regulates ARD initiation through a pathway involving the O-GlcNAc cycling enzyme, OGT-1, acting with the nuclear hormone receptor NHR-49. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings thus identify novel conserved genetic pathways required for ARD entry and recovery and identify new genetic interactions that provide insight into the role of OGT and OGA.

genetics↗

bioRxiv2019

Microbiome Determinants and Physiological Effects of the Benzoate-Hippurate Microbial-Host Co-Metabolic Pathway

ObjectiveGut microbial products are involved in type 2 diabetes, obesity and insulin resistance. In particular, hippurate, a hepatic phase 2 conjugation product of microbial benzoate metabolism, has been associated with a healthy phenotype. This study aims to identify metagenomic determinants and test protective effects of hippurate. DesignWe profiled the urine metabolome by 1H Nuclear Magnetic Resonance (NMR) spectroscopy to derive associations with metagenomic sequences in 271 middle-aged Danish individuals to identify dietary patterns in which urine hippurate levels were associated with health benefits. We follow up with benzoate and hippurate infusion in mice to demonstrate causality on clinical phenotypes. ResultsIn-depth analysis identifies that the urine hippurate concentration is associated with microbial gene richness, microbial functional redundancy as well as functional modules for microbial benzoate biosynthetic pathways across several enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate, independently of gene richness, accounts for links with metabolic health that we previously associated with gene richness. We then demonstrate causality in vivo through chronic subcutaneous infusions of hippurate or benzoate (20 nmol/day) resulting in improved glycemic control in mice fed a high-fat diet. Hippurate improved insulin secretion through increased {beta}-cell mass and reduced liver inflammation and fibrosis, whereas benzoate treatment resulted in liver inflammation. ConclusionOur translational study shows that the benzoate-hippurate pathway brings a range of metabolic improvements in the context of high-fat diets, highlighting the potential of hippurate as a mediator of metabolic health.

systems biology↗

bioRxiv2019

Towards the molecular architecture of the peroxisomal receptor docking complex

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles are however unclear, mainly because structures of higher order assemblies are still lacking. Here, using an integrative approach, we provide the first structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20 nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding-sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

molecular biology↗