Biology subjects

Johannes, L.

Publications and source records attributed to Johannes, L..

13 recordsLinked to original sources

bioRxiv2026

Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

Aberrant endocytosis has long been associated with epithelial plasticity and tumorigenesis, but direct in vivo evidence of its causal role in tumor progression and metastasis has been lacking. Here, we identify and molecularly characterize a previously unrecognized form of E-cadherin (ECAD) internalization in mammary epithelial cells. This process is mediated by the endocytic adaptor Epsin 3 (EPN3) through glycolipid-lectin (GL-Lect) driven endocytosis requiring galectin-3 and Eps15-family adaptors. Leveraging an EPN3 knock-in mouse model, we show that dysregulation of GL-Lect driven endocytosis disrupts mammary gland morphogenesis and activates epithelial-to-mesenchymal plasticity (EMP), synergizing with the ERBB2/Neu breast oncogene to drive metastasis. Pharmacologic inhibition of the GL-Lect mechanism suppresses morphogenetic and invasive phenotypes ex vivo, providing proof-of-concept for therapeutic targeting. These findings establish the GL-Lect mechanism as a driver of metastatic plasticity and uncover a tractable vulnerability in BC.

cancer biology↗

bioRxiv2025

Galectin-3 Mediated Endocytosis of the Orphan G-Protein-Coupled Receptor GPRC5A

Galectins, a family of glycan-binding proteins, play crucial roles in various cellular functions, acting at both intracellular and extracellular levels. Among them, Galectin-3 (Gal-3) stands out as a unique member, possessing an intrinsically disordered N-terminal interaction domain and a canonical carbohydrate-recognition domain (CRD). Gal-3 binding to glycosylated plasma membrane cargo leads to its oligomerization and membrane bending, ultimately resulting in the formation of endocytic invaginations. An interactomic assay using proteomic analysis of endogenous Gal-3 immunoprecipitates identified the orphan G protein-coupled receptor GPRC5A as a novel binding partner of Gal-3. GPRC5A, also known as Retinoic Acid-Induced protein 3 (RAI3), is transcriptionally induced by retinoic acid. Our results further demonstrate that extracellular recombinant Gal-3 stimulates GPRC5A internalization. In SW480 colorectal cancer cells, glycosylated GPRC5A interacts with Gal-3. Interestingly, while GPRC5A expression was upregulated by the addition of all-trans retinoic acid (ATRA), its endogenous internalization in SW480 cells was specifically triggered by extracellular Gal-3, but not by ATRA. This study provides new insights into the endocytic mechanisms of GPRC5A, for which no specific ligand has been identified to date. Further research may uncover additional Gal-3 mediated functions in GPRC5A cellular signaling and contribute to the development of innovative therapeutic strategies.

cell biology↗

bioRxiv2024

Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

Endophilin A3-mediated clathrin-independent endocytosis (EndoA3-mediated CIE) contributes to the internalization of immunoglobulin-like proteins, including key immune synapse components. Here, we identify ICAM1 as a novel EndoA3-dependent cargo, alongside ALCAM. We demonstrate that both proteins subsequently follow retromer-dependent retrograde transport to the trans-Golgi network (TGN) in cancer cells. From there, we propose that they undergo polarized redistribution to the plasma membrane, where they contribute to immune synapse formation between cancer cells and cytotoxic CD8 T cells. Disruption of EndoA3 or retromer components significantly affects the response of autologous cytotoxic CD8 T cells, as evidenced by reduced cytokine production and secretion, but increased lytic activity, while proliferation and later activation marker expression remain intact. This is accompanied by diminished ICAM1 density at the immune synapse, where we observe it arriving via polarized vesicular transport, indicating altered synapse organization. Indeed, cancer cells lacking EndoA3-mediated CIE or retromer form enlarged immune synapses that fail to sustain full T cell cytokine secretion, suggesting a compensatory attempt by T cells to overcome the defective synapse, while likely promoting more transient contacts that potentially favor serial killing. Together, these findings reveal that EndoA3-mediated CIE and retrograde transport act in concert in cancer cells to relocate immune synapse components via the Golgi, thereby fine-tuning the balance between cytotoxic T cell cytokine secretion and lytic activity. These insights contribute to a better understanding of the mechanisms governing immune synapse formation and organization, providing a necessary foundation for the long-term identification of new strategies to enhance T cell-mediated anti-tumor immunity. Significance StatementThis study uncovers a novel mechanism by which clathrin-independent endocytosis (CIE) and retrograde transport collaborate to regulate immune synapse dynamics in cancer cells. We identify ICAM1 as a new cargo of Endophilin A3-mediated CIE, highlighting its role in the polarized redistribution of immune synapse components critical for cytotoxic CD8 T cell activation. By linking a specific CIE mechanism and retrograde transport to immune synapse function, our findings provide new insights into cancer-immunity interactions and suggest potential therapeutic strategies to enhance immune responses by targeting protein trafficking pathways.

cell biology↗

bioRxiv2024

Novel, postnatal manifestation of an epidermal barrier defect in a mouse model of isolated sulfite oxidase deficiency

Sulfite oxidase deficiency is a rare inborn error in metabolism leading to early childhood death due to rapidly progressing neurodegeneration. A new mouse model of sulfite oxidase deficiency carrying a homozygous deletion in the Suox gene resembles the human pathology in terms of neonatal death and elevation of sulfite and thiosulfate in plasma and urine, respectively. Homozygous Suox-/- mice are initially born healthy, display growth retardation starting at postnatal day 4 and die in average at day 9.6. Here we report that Suox-/- mice develop dry and scaly skin early postnatally, showing that sulfite oxidase is essential to maintain a functional skin barrier after birth. At postnatal day 5 Suox-/- mice develop altered epidermal morphology and dysregulated early and late keratinocyte differentiation accompanied by increased stress response. We propose a sulfite-induced cleavage of disulfide bonds in key epidermal proteins essential for a functional barrier.

biochemistry↗

bioRxiv2024

Differential predictive value of resident memory CD8+T cell subpopulations in non-small-cell lung cancer patients treated by immunotherapy

A high density of resident memory T cells (TRM) in tumors correlates with improved clinical outcomes in immunotherapy-treated patients. However, in preclinical models, only some subpopulations of TRM are associated with cancer vaccine efficacy. We identified two main TRM subpopulations in tumor-infiltrating lymphocytes derived from non-small cell lung cancer (NSCLC) patients: one co-expressing CD103 and CD49a (DP), and the other expressing only CD49a (MP); both exhibiting additional TRM surface markers like CD69. DP TRM exhibited greater functionality compared to MP TRM. Analysis of T-cell receptor (TCR) repertoire and of the stemness marker TCF-1 revealed shared TCRs between populations, with the MP subset appearing more progenitor-like phenotype. In two NSCLC patient cohorts, only DP TRM predicted PD-1 blockade response. Multivariate analysis, including various biomarkers (CD8, TCF1+CD8+T cells, and PD-L1) associated with responses to anti-PD(L)1, showed that only intra-tumoral infiltration by DP TRM remained significant. This study highlights the non-equivalence of TRM populations and emphasizes the importance of distinguishing between them to better define their role in antitumor immunity and as a biomarker of response to immunotherapy.

immunology↗

bioRxiv2023

Spatial N-glycan rearrangement on α5β1 integrin nucleates galectin-3 oligomers to determine endocytic fate α

Membrane glycoproteins frequently adopt different conformations when altering between active and inactive states. Here, we discover a molecular switch that exploits dynamic spatial rearrangements of N-glycans during such conformational transitions to control protein function. For the conformationally switchable cell adhesion glycoprotein 5{beta}1 integrin, we find that only the bent-closed state arranges N-glycans to nucleate the formation of up to tetrameric oligomers of the glycan-binding protein galectin-3. We propose a structural model of how these galectin-3 oligomers are assembled and how they clamp the bent-closed state to prime it for endocytic uptake and subsequent retrograde trafficking to the Golgi for polarized distribution in cells. Our findings highlight an unexpectedly dynamic regulation of the glycan landscape at the cell surface to achieve oligomerization of galectin-3. Galectin-3 oligomers are thereby identified as decoders of defined spatial patterns of N-glycans and as functional extracellular interactors of specifically the bent- closed conformational state of 5{beta}1 integrin and possibly other family members.

cell biology↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2023

A synthetic delivery vector for mucosal vaccination

The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.

biochemistry↗

bioRxiv2022

SLC3A2 N-glycosylation and alternate evolutionary trajectories for amino acid metabolism

SLC3A2 (4F2hc, CD98) is an adaptor to the SLC7A exchangers and has undergone extensive repositioning of N-glycosylation sites with vertebrate evolution, presumably in synchrony with the species-specific demands of metabolism. The SLC3A2*SLC7A5 heterodimer imports essential amino acids (AA) and thereby stimulates mTOR signaling, while SLC3A2*SLC7A11 imports cystine required for glutathione synthesis and mitigation of oxidative stress. Analysis of SLC3A2 N-glycans revealed stable site-specific profiles of Golgi remodeling, apart from the conserved N365 site where branching and poly-N-acetylglucosamine content were sensitive to the insertion of lost ancestral sites and to metabolism. N-glycans at N381 and N365 stabilized SLC3A2 in the galectin lattice and opposed endocytosis, while N365 which is nearest the membrane, also promoted down-regulation by galectin-driven clathrin-independent endocytosis (glycolipid-lectin GL-Lect). This is the first report of both positive and negative regulation by galectin binding to N-glycans that are strategically positioned in the same membrane glycoproteins. Proteomics analysis in SLC3A2 mutant HeLa cells with induced re-expression of SLC3A2 as bait revealed the canonical non-N-glycosylated interactors, SLC7A5 and SLC7A11 exchangers, but also AA transporters that were dependent on SLC3A2 N-glycosylation, and are themselves, N-glycosylated AA/Na+ symporters (SLC7A1, SLC38A1, SLC38A2, SLC1A4, SLC1A5). The results suggest that the N-glycans on SLC3A2 regulate clustering of SLC7A exchangers with AA/Na+ symporters, thereby promoting Gln/Glu export-driven import of essential AA and cystine, with the potential to adversely impact redox balance. The evolution of modern birds (Neoaves) led to improved control of bioenergetics with the loss of genes including SLC3A2, SLC7A-5, -7, -8, -10, BCAT2, KEAP1, as well as duplications of SLC7A9, SLC7A11 and the Golgi branching enzymes MGAT4B and MGAT4C known to enhance affinities for galectins. Analyzing the fate of these and other genes in the down-sized genomes of birds, spanning [~]10,000 species and >100 Myr of evolution, may reveal the mystery of their longevity with prolonged vitality. Key PointsO_LIGolgi N-glycan remodeling at each site on SLC3A2 differs with the microenvironment. C_LIO_LIThe galectin lattice and GL-Lect mediated endocytosis act as opposing forces on trafficking, controlled by N-glycans at the distal N381 and membrane proximal N365 sites, respectively. C_LIO_LIMutation at N381 or N365 decreased SLC3A2 association with SLC7A5, SLC7A11 and N-glycosylated AA/Na+ symporters as well as the capacity to mitigate stress. C_LIO_LIClustering of SLC3A2*SLC7A exchangers, with AA/Na+ symporter and ATPase Na+/K+ exchanger promotes growth but continuously consumes ATP in non-proliferating cells. C_LIO_LIBird evolution has improved bioenergetics with the deletion of SLC3A2 and associated transporters; - replaced by transporters of keto acids and a re-enforced galectin lattice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/516651v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1cb7958org.highwire.dtl.DTLVardef@18ba32dorg.highwire.dtl.DTLVardef@861269org.highwire.dtl.DTLVardef@22df0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

microbiology↗

bioRxiv2020

Retrograde And Anterograde Transport Of LAT-Vesicles During The Immunological Synapse Formation: Defining The Finely-Tuned Mechanism

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T-cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of 4 proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute in both pathways, are in our cellular context specifically and respectively involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

cell biology↗

bioRxiv2019

STAM Interaction with Hrs Controls JAK/STAT Activation by Interferon-α at the Early Endosome

Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway by type I interferons (IFN) requires clathrin-dependent endocytosis of the IFN-/{beta} receptor (IFNAR). The molecular machinery that brings about the selective activation of IFN-/{beta}-induced JAK/STAT signaling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and the TYK2 Janus kinase at the plasma membrane prevents the activation of TYK2 by type I IFNs. IFN- stimulated endocytosis leads to the interaction of IFNAR1 with Hrs on early endosomes, which then relieves TYK2 inhibition by STAM and thereby allows for TYK2 and IFNAR signaling. In contrast, IFN-{beta} stimulation results in sorting of IFNAR to a distinct endosomal subdomain where the receptor is activated independently from Hrs. Our results identify the molecular machinery that controls the spatiotemporal activation of TYK2 and establish the central role of endosomal sorting in the differential regulation of JAK/STAT signaling by IFN- and IFN-{beta}.\n\nSummaryThe spatiotemporal activation of JAK/STAT signaling by IFN- is controlled by STAM association with Hrs at the early endosome.

cell biology↗

bioRxiv2018

Lack of functional caveolae in Cav3 mutated human dystrophic myotubes results in deficient mechanoprotection and IL6/STAT3 mechanosignaling.

Caveolin-3 is the major structural protein of caveolae in muscle cells. Mutations in the CAV3 gene cause different type of muscle disorders mostly characterized by defects in membrane integrity and repair, deregulation in the expression of various muscle proteins and deregulation of several muscle associated signaling pathways. We show here that myotubes derived from patients bearing the CAV3 P28L and R26Q mutations present a lack of functional caveolae at the plasma membrane which results in an abnormal mechanoresponse. Mutant myotubes can no longer buffer the increase of membrane tension induced by mechanical stress and present an hyperactivation of the IL6/STAT3 signaling pathway at rest and under mechanical stress. The impaired mechanical regulation of the IL6/STAT3 signaling pathway by caveolae leads to chronic activation and a higher expression of muscle specific genes. These defects could be reversed by reassembling a pool of functional caveolae through expression of wild type Cav3. Our findings bring more mechanistic insight into human Cav3 associated muscle disorders and show a general defect in the mechanoresponse of CAV3 P28L and R26Q myotubes.

cell biology↗