PSL Chemical Biology Symposia: Recent Progress in Ferroptosis.

This symposium is the 5th PSL (Paris Sciences & Lettres) Chemical Biology meeting (2015, 2016, 2019, 2023, 2024) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition hosted around 150 participants and was focused on the burgeoning field of ferroptosis, its mechanism and implications in health and disease. While not initially planned, it was felt that the next large Ferroptosis venue (CSHA, China) would not happen before late 2024. A discussion involving Conrad, Birsoy, Ubellacker, Brabletz and Rodriguez next to lake Como in Italy sponsored by the DKFZ, prompted us to fill in this gap and to organize a Ferroptosis meeting in Paris beforehand.

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

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.

Engineered Synthetic STxB for Enhanced Cytosolic Delivery.

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8+ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Solubilization and Purification of α5ß1 Integrin from Rat Liver for Reconstitution into Nanodiscs.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α5ß1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5ß1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

STxB as an Antigen Delivery Tool for Mucosal Vaccination.

Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4+ and CD8+ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8+ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer.

The roles of dynein and myosin VI motor proteins in endocytosis.

Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.

High-Relaxivity Molecular MRI Contrast Agent to Target Gb3-Expressing Cancer Cells.

Targeted contrast agents (CAs) can improve magnetic resonance imaging (MRI) for accurate cancer diagnosis. In this work, we used the Shiga toxin B-subunit (STxB) as a targeting agent, which binds to Gb3, a glycosphingolipid highly overexpressed on the surface of tumor cells. We developed STxB-targeted MRI probes from cyclic peptide scaffolds functionalized with six to nine monoamide DO3A[Gd(III)] chelates. The influence of structural constraints on the longitudinal relaxivity (r1) of the CAs has been studied. The cyclic peptide carrying nine monoamide DO3A[Gd(III)] exhibited a r1 per compound of 32 and 93 mM-1s-1 at 9.4 and 1.5 T, respectively. Its conjugation to the pentameric STxB protein led to a 70 kDa compound with a higher r1 of 150 and 475 mM-1 s-1 at 9.4 and 1.5 T, respectively. Specific accumulation and cellular distribution of this conjugate in Gb3-expressing cancer cells were demonstrated using immunofluorescence microscopy and quantified by an inductively coupled plasma-mass spectrometry dosage of Gd(III). Such an agent should enable the in vivo detection by MRI of tumors expressing Gb3 receptors.

Tumor Targeting with Bacterial Shiga Toxin B Subunit in Genetic Porcine Models for Colorectal Cancer and Osteosarcoma.

The B subunit of bacterial Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the tumor-targeting potential of STxB. In accordance with findings on human colorectal cancer, the pig model APC1311 bound STxB in colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for osteosarcoma, a tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig osteosarcoma strongly bound StxB and expressed the Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human osteosarcoma cell lines expressed A4GALT mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based tumor targeting and may allow "reverse-translational" predictions on human tumor biology.

The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery-The GL-Lect Hypothesis.

Lipid membranes are common to all forms of life. While being stable barriers that delimitate the cell as the fundamental organismal unit, biological membranes are highly dynamic by allowing for lateral diffusion, transbilayer passage via selective channels, and in eukaryotic cells for endocytic uptake through the formation of membrane bound vesicular or tubular carriers. Two of the most abundant fundamental fabrics of membranes-lipids and complex sugars-are produced through elaborate chains of biosynthetic enzymes, which makes it difficult to study them by conventional reverse genetics. This review illustrates how organic synthesis provides access to uncharted areas of membrane glycobiology research and its application to biomedicine. For this Special Issue on Chemical Biology Research in France, focus will be placed on synthetic approaches (i) to study endocytic functions of glycosylated proteins and lipids according to the GlycoLipid-Lectin (GL-Lect) hypothesis, notably that of Shiga toxin; (ii) to mechanistically dissect its endocytosis and intracellular trafficking with small molecule; and (iii) to devise intracellular delivery strategies for immunotherapy and tumor targeting. It will be pointed out how the chemical biologist's view on lipids, sugars, and proteins synergizes with biophysics and modeling to "look" into the membrane for atomistic scale insights on molecular rearrangements that drive the biogenesis of endocytic carriers in processes of clathrin-independent endocytosis.

CXCR6 deficiency impairs cancer vaccine efficacy and CD8+ resident memory T-cell recruitment in head and neck and lung tumors.

BACKGROUND: Resident memory T lymphocytes (TRM) are located in tissues and play an important role in immunosurveillance against tumors. The presence of TRM prior to treatment or their induction is associated to the response to anti-Programmed cell death protein 1 (PD-1)/Programmed death-ligand 1 (PD-L1) immunotherapy and the efficacy of cancer vaccines. Previous work by our group and others has shown that the intranasal route of vaccination allows more efficient induction of these cells in head and neck and lung mucosa, resulting in better tumor protection. The mechanisms of in vivo migration of these cells remains largely unknown, apart from the fact that they express the chemokine receptor CXCR6. METHODS: We used CXCR6-deficient mice and an intranasal tumor vaccination model targeting the Human Papillomavirus (HPV) E7 protein expressed by the TC-1 lung cancer epithelial cell line. The role of CXCR6 and its ligand, CXCL16, was analyzed using multiparametric cytometric techniques and Luminex assays.Human biopsies obtained from patients with lung cancer were also included in this study. RESULTS: We showed that CXCR6 was preferentially expressed by CD8+ TRM after vaccination in mice and also on intratumoral CD8+ TRM derived from human lung cancer. We also demonstrate that vaccination of Cxcr6-deficient mice induces a defect in the lung recruitment of antigen-specific CD8+ T cells, preferentially in the TRM subsets. In addition, we found that intranasal vaccination with a cancer vaccine is less effective in these Cxcr6-deficient mice compared with wild-type mice, and this loss of efficacy is associated with decreased recruitment of local antitumor CD8+ TRM. Interestingly, intranasal, but not intramuscular vaccination induced higher and more sustained concentrations of CXCL16, compared with other chemokines, in the bronchoalveolar lavage fluid and pulmonary parenchyma. CONCLUSIONS: This work demonstrates the in vivo role of CXCR6-CXCL16 axis in the migration of CD8+ resident memory T cells in lung mucosa after vaccination, resulting in the control of tumor growth. This work reinforces and explains why the intranasal route of vaccination is the most appropriate strategy for inducing these cells in the head and neck and pulmonary mucosa, which remains a major objective to overcome resistance to anti-PD-1/PD-L1, especially in cold tumors.

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse.

BACKGROUND: Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. METHODS: We designed a mouse model of tumor relapse in which mice transplanted with E7+ TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. RESULTS: We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. CONCLUSIONS: Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

Glycosylation and raft endocytosis in cancer.

Changes in glycosylation on proteins or lipids are one of the hallmarks of tumorigenesis. In many cases, it is still not understood how glycan information is translated into biological function. In this review, we discuss at the example of specific cancer-related glycoproteins how their endocytic uptake into eukaryotic cells is tuned by carbohydrate modifications. For this, we not only focus on overall uptake rates, but also illustrate how different uptake processes-dependent or not on the conventional clathrin machinery-are used under given glycosylation conditions. Furthermore, we discuss the role of certain sugar-binding proteins, termed galectins, to tune glycoprotein uptake by inducing their crosslinking into lattices, or by co-clustering them with glycolipids into raft-type membrane nanodomains from which the so-called clathrin-independent carriers (CLICs) are formed for glycoprotein internalization into cells. The latter process has been termed glycolipid-lectin (GL-Lect) hypothesis, which operates in a complementary manner to the clathrin pathway and galectin lattices.

MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue.

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling.

Galectin-3 (Gal3) is a multifaceted protein which belongs to a family of lectins and binds ß-galactosides. Gal3 expression is altered in many types of cancer, with increased expression generally associated with poor prognosis. Although the mechanisms remain unknown, Gal3 has been implicated in several biological processes involved in cancer progression, including suppression of T cell-mediated immune responses. Extracellular Gal3 binding to the plasma membrane of T cells alters membrane organization and the formation of an immunological synapse. Its multivalent capacity allows Gal3 to interact specifically with different membrane proteins and lipids, influencing endocytosis, trafficking and T cell receptor signalling. The ability of Gal3 to inhibit T cell responses may provide a mechanism by which Gal3 aids in cancer progression. In this review, we seek to give an overview of the mechanisms by which Gal3 alters the spatial organization of cell membranes and how these processes impact on T cell activation.

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells.

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.