Publications by authors named "Anne Billet"

8 Publications

  • Page 1 of 1

Absolute Quantification of Drug Vector Delivery to the Cytosol.

Angew Chem Int Ed Engl 2021 Jun 28;60(27):14824-14830. Epub 2021 May 28.

Cellular and Chemical Biology Unit, U1143 INSERM, UMR3666 CNRS, Institut Curie-, Université PSL, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.
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http://dx.doi.org/10.1002/anie.202102332DOI Listing
June 2021

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

Toxins (Basel) 2020 07 10;12(7). Epub 2020 Jul 10.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden.

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.
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http://dx.doi.org/10.3390/toxins12070449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404996PMC
July 2020

Glycosylation and raft endocytosis in cancer.

Cancer Metastasis Rev 2020 06;39(2):375-396

Cellular and Chemical Biology Unit, INSERM U1143, CNRS UMR3666, Institut Curie, PSL Research University, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

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.
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http://dx.doi.org/10.1007/s10555-020-09880-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311491PMC
June 2020

An underestimated sexually transmitted infection: amoebiasis.

BMJ Case Rep 2019 May 10;12(5). Epub 2019 May 10.

Infectious diseases, Centre Hospitalier Universitaire Dijon-Bourgogne, Dijon, France.

is a cosmopolitan pathogenic parasite. It is spread via the feco-oral route and, to a lesser extent, via sexual intercourse. We report a case of hepatic and intestinal amoebiasis in a 67-year-old man who had never travelled to an endemic area. Abdominal CT investigations detected two liver abscesses and chronic colitis. Positive amoebic serology and a positive PCR test for in the hepatic liquid and faeces confirmed the diagnosis. Curative metronidazole and tiliquinol-tilbroquinol were administered successfully. The patient had been contaminated through heterosexual intercourse with his healthy French female partner who was a carrier of the parasite. Though unusual, amoebiasis as a result of sexual transmission should be considered in non-endemic areas in people who have never travelled abroad, particularly in the presence of clinical symptoms such as liver abscesses or chronic diarrhoea.
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http://dx.doi.org/10.1136/bcr-2018-228942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536245PMC
May 2019

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chembiochem 2019 04 25;20(7):968-973. Epub 2019 Feb 25.

PSL Université Paris, Institut Curie, CNRS UMR3666, INSERM U1143, 75005, Paris, France.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.
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http://dx.doi.org/10.1002/cbic.201900092DOI Listing
April 2019

Rosetta FunFolDes - A general framework for the computational design of functional proteins.

PLoS Comput Biol 2018 11 19;14(11):e1006623. Epub 2018 Nov 19.

Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

The robust computational design of functional proteins has the potential to deeply impact translational research and broaden our understanding of the determinants of protein function and stability. The low success rates of computational design protocols and the extensive in vitro optimization often required, highlight the challenge of designing proteins that perform essential biochemical functions, such as binding or catalysis. One of the most simplistic approaches for the design of function is to adopt functional motifs in naturally occurring proteins and transplant them to computationally designed proteins. The structural complexity of the functional motif largely determines how readily one can find host protein structures that are "designable", meaning that are likely to present the functional motif in the desired conformation. One promising route to enhance the "designability" of protein structures is to allow backbone flexibility. Here, we present a computational approach that couples conformational folding with sequence design to embed functional motifs into heterologous proteins-Rosetta Functional Folding and Design (FunFolDes). We performed extensive computational benchmarks, where we observed that the enforcement of functional requirements resulted in designs distant from the global energetic minimum of the protein. An observation consistent with several experimental studies that have revealed function-stability tradeoffs. To test the design capabilities of FunFolDes we transplanted two viral epitopes into distant structural templates including one de novo "functionless" fold, which represent two typical challenges where the designability problem arises. The designed proteins were experimentally characterized showing high binding affinities to monoclonal antibodies, making them valuable candidates for vaccine design endeavors. Overall, we present an accessible strategy to repurpose old protein folds for new functions. This may lead to important improvements on the computational design of proteins, with structurally complex functional sites, that can perform elaborate biochemical functions related to binding and catalysis.
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http://dx.doi.org/10.1371/journal.pcbi.1006623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277116PMC
November 2018

Natural mutations in IFITM3 modulate post-translational regulation and toggle antiviral specificity.

EMBO Rep 2016 11 6;17(11):1657-1671. Epub 2016 Sep 6.

Virus & Immunity Unit, Institut Pasteur, Paris, France

The interferon-induced transmembrane (IFITM) proteins protect host cells from diverse virus infections. IFITM proteins also incorporate into HIV-1 virions and inhibit virus fusion and cell-to-cell spread, with IFITM3 showing the greatest potency. Here, we report that amino-terminal mutants of IFITM3 preventing ubiquitination and endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. This suggests that "runaway" IFITM3 variants could be selected for altered antiviral activity. Furthermore, we show that adaptations in IFITM3 result in a trade-off in antiviral specificity, as variants exhibiting enhanced activity against HIV-1 poorly restrict influenza A virus. Overall, we provide the first experimental evidence that diversification of IFITM3 genes may boost the antiviral coverage of host cells and provide selective functional advantages.
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http://dx.doi.org/10.15252/embr.201642771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090704PMC
November 2016

Refractory venous leg ulcers: a study of risk factors.

Dermatol Surg 2006 Apr;32(4):512-9

Department of Dermatology, Hôpital Sud, Amiens, France.

Background: Although certain risk factors for poor healing of leg ulcers have been identified, data concerning the characteristics of refractory ulcers have not been specifically studied in the literature.

Objective: To study the characteristics of refractory venous leg ulcers.

Methods: We retrospectively studied prognostic factors for healing in patients with refractory venous leg ulcers followed and treated in our dermatology department between January 1993 and January 2000. Each patient included in this study was compared with two patients matched for age and gender and presenting leg ulcers with normal healing, followed during the same period.

Results: Thirty-two of 571 patients with leg ulcers were included. The study population consisted of 20 females and 12 males with a mean age 73.5 years. The control population comprised 64 patients, 40 females and 24 males, with a mean age of 73 years. Univariate analysis demonstrated the negative prognostic impact of several previously identified factors (including surface area and history of the ulcer). In particular, multivariate analysis identified four main risk factors for refractory ulcer that are often associated in these patients: associated arterial disease, presence of post-thrombotic popliteal sequelae, recurrence of the ulcer, and disability.

Conclusion: Four main risk factors that are often associated were identified, indicating the multifactorial nature of these refractory ulcers.
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http://dx.doi.org/10.1111/j.1524-4725.2006.32104.xDOI Listing
April 2006