Biology subjects

Roemer, W.

Publications and source records attributed to Roemer, W..

9 recordsLinked to original sources

bioRxiv2026

Fibroblast-derived Collagen VI shapes the structure and function of the tumor-immune microenvironment in clear cell renal cell carcinoma

The importance of the extracellular matrix (ECM) influencing tumor biology in stroma-rich tumors is well established. However, the relevance of individual ECM proteins in rather stroma-poor cancers such as clear cell renal cell carcinoma (ccRCC) is ill-defined. Using bulk proteomics, spatial imaging, and single-cell transcriptomics, we identify collagen VI (COL6) as a predominant ECM component of the ccRCC interstitial stroma, synthesized primarily by fibroblasts and pericytes. Using cell-derived matrix (CDM) models, we demonstrate that COL6 is essential for maintaining an isotropic ECM network architecture and governs the broader matrisomal composition, with direct pro-proliferative consequences for tumor cells both in vitro and in situ. Granular spatial analysis reveals that COL6-rich stromal septa constrain tumor-infiltrating T cells to boundary zones, where CD8+PD1+ phenotypes predominate. Importantly, tyrosine kinase inhibition (TKI) with cabozantinib suppresses COL6 expression in fibroblasts in vitro and in ex vivo tumor models, mirroring COL6-depleted CDM phenotypes. Our findings establish COL6 as a central stromal regulator of ccRCC tumor biology and immune contexture, revealing ECM remodeling as an underappreciated mechanism of TKI action, with implications for combination immunotherapy strategies.

cancer biology↗

bioRxiv2023

Real-time monitoring of cell surface protein arrival with split luciferases

Each cell in a multicellular organism permanently adjusts the concentration of its cell surface proteins. In particular, epithelial cells tightly control the number of carriers, transporters and cell adhesion proteins at their plasma membrane. However, the sensitively measuring the cell surface concentration of a particular protein of interest in live cells and in real time represents a considerable challenge. Here, we introduce a novel approach based on split luciferases, which uses one luciferase fragment as tag on the protein of interest and the second fragment as supplement to the extracellular medium. Once the protein of interest arrives at the cell surface, the luciferase fragments complement and generate luminescence. We compared the performance of split Gaussia luciferase and split Nanoluciferase by using a system to synchronize biosynthetic trafficking with conditional aggregation domains. The best results were achieved with split Nanoluciferase, for which luminescence increased more than 6000-fold upon recombination. Furthermore, we showed that our approach can separately detect and quantify the arrival of membrane proteins at the apical and basolateral plasma membrane in single polarized epithelial cells by detecting the luminescence signals with a microscope, thus opening novel avenues for characterizing the variations in trafficking in individual epithelial cells. SynopsisWe present here a novel method to determine the arrival of a membrane protein of interest at the cell surface in real time and with sufficient sensitivity to achieve single-cell resolution. This allows measuring detailed kinetics of plasma membrane trafficking and thus to detect also minor changes in protein sorting and intracellular trafficking. Furthermore, the single-cell sensitivity of the method will enable to systematically characterize variations in trafficking between neighboring cells within a multicellular organism.

cell biology↗

bioRxiv2022

Extending Janus lectins architecture: characterization and application to protocells

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being developed for producing tools with precise topology and specificity. We developed the concept of chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the {beta}-propeller domain from Ralstonia solanacearum and the {beta}-trefoil domain from fungus Marasmius oreades, is specific for fucose and -galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The synthetic protein binds to H1299 lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

synthetic biology↗

bioRxiv2022

The bacterial lectin LecA from P. aeruginosa alters membrane organization by dispersing ordered domains

The assembly and dynamic reorganization of plasma membrane nanodomains (also known as "lipid rafts") play key roles in host cell infection by human pathogens (e.g. viruses and bacteria). Viruses and bacteria can trigger the reorganization of lipid rafts which leads to membrane invaginations and downstream signaling that promote infection. Such reorganizations can be induced by interactions of bacterial or viral carbohydrate proteins (so-called lectins) with lipid raft glycosphingolipids (GSLs). Here, we studied the GSL globotriaosylceramide (Gb3) which is a key receptor involved in the cellular uptake of the gram-negative bacterium P. aeruginosa. The bacterial surface lectin LecA targets Gb3 and promotes bacterial invasion via the "lipid zipper" mechanism. However, the impact of LecA on the organization of membrane nanodomains is unknown yet. We mimicked of the plasma membrane using supported lipid bilayers (SLBs) that contained liquid-ordered (Lo, "raft-like", enriched in sphingolipids and GSLs) and liquid-disordered (Ld, "non-raft-like" enriched in DOPC) lipid domains. Upon interaction with LecA, the Lo domains in the SLBs reshaped and dispersed. Moreover, deformation of SLBs was observed as LecA formed membrane multilayers on SLBs surface. We further dissected this process to reveal the impact of Gb3 structure, bilayer composition and LecA valence on the Lo reorganization.

biophysics↗

bioRxiv2022

A pore-forming β-trefoil lectin with specificity for the tumor-related glycosphingolipid Gb3

Lectins are efficient multivalent glycan receptors, deciphering the glyco-code on cell surfaces. The {beta}-trefoil fold, characterized by three lobe-shaped repeats, is adopted by several classes of lectins, often associated with other domains having enzymatic or toxic activity. Based on the UniLectin3D database classification, the sequence signature of trefoil lobes was defined and used to predict 44714 lectins from 4497 species. Among them, SaroL-1 from the lower eukaryote Salpingoeca rosetta was predicted to contain both {beta}-trefoil and aerolysin-like pore-forming domain. Recombinant SaroL-1 binds to galactose and derivatives, with a stronger affinity for cancer-related -galactosylated epitopes such as glycosphingolipid Gb3 embedded in giant unilamellar vesicles or cell membranes. Crystal structures in complex with Gb3 trisaccharide and GalNAc show similarity with pore-forming toxins. Recognition of the Gal epitope on glycolipids was necessary for hemolysis of rabbit erythrocytes and toxicity on cancer cells through carbohydrate-dependent pore-formation.

biochemistry↗

bioRxiv2021

Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of endorgan damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that longterm use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated -Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

molecular biology↗

bioRxiv2020

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a high number of acute and chronic hospital-acquired infections. As it develops more and more resistances against existing antibiotics, P. aeruginosa has been placed highest on the global priority list of antibiotic-resistant bacteria for which alternative treatments are urgently needed. Former studies have highlighted the crucial role of the bacterial lectin LecA and the host cell glycosphingolipid globotriaosylceramide (Gb3) for the cellular uptake of P. aeruginosa into epithelial cells via the lipid zipper mechanism. To further characterize the host cell plasma membrane domain for LecA-driven attachment and invasion, we analyzed the protein and lipid composition of pulled-down membrane domains for novel interaction partners of LecA by mass spectrometry. We unraveled a predilection of LecA for binding to saturated Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate clusters at the intracellular leaflet co-localizing with sites of LecA binding. Moreover, we identified the GPI-anchored protein CD59 and flotillins, known as cargo and eponymous component of flotillin-assisted endocytosis, as LecA interaction partners. Depletion of each of these host cell proteins resulted in more than 50% of reduction in invasiveness of the P. aeruginosa strain PAO1 highlighting the importance of this LecA-induced plasma membrane domain. Our strategy to reduce the complexity of host-pathogen interactions by first identifying interaction partners of a single virulence factor and subsequently transferring these findings to the bacterium has been proven to be a successful approach in elucidating the molecular mechanisms of bacterial infections.

cell biology↗

bioRxiv2019

The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as crosslinker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of {beta}1-integrins on the basolateral plasma membrane of epithelial cells and caused rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive {beta}1-integrins and also of complete 3{beta}1-integrin - laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and caused the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by crosslinking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results we suggest that LecB has a role in disseminating bacteria along the cell - basement membrane interface. ImportancePseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes for nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interaction is not well understood. The significance of our research is in identifying cellular mechanisms how LecB facilitates P. aeruginosa infection: We introduce LecB as new member to the list of bacterial molecules that bind integrins and show that P. aeruginosa can efficiently move forward underneath attached epithelial cells by loosening cell - basement membrane attachment in a LecB-dependent manner.

microbiology↗

bioRxiv2019

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria employ lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection.\n\nHere, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, that extend beyond its role as an adhesion molecule. We found that LecB associates with several growth factor receptors and dampens their signalling pathways, leading to the arrest of cell cycle. Additionally, we describe a novel LecB-triggered mechanism to downregulate host cell receptors by showing that LecB leads to insulin-like growth factor receptor 1 internalisation, without receptor activation, and subsequent missorting towards intracellular endosomal compartments.\n\nOverall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

cell biology↗