Laetitia Coassolo (Voilquin)

All Publications

• ANGPTL3 orchestrates hepatic fructose sensing and metabolism. Cell reports Zhao, M., Linde-Garelli, K. Y., Zhang, Z., Toomer, D., Reghupaty, S. C., Jimenez, J. I., Coassolo, L., Wat, L. W., Fernandez, D., Svensson, K. J. 2025; 44 (7): 115962

  Abstract

  Fructose metabolism is linked to metabolic dysfunction-associated steatotic liver disease (MASLD), but the regulatory mechanisms governing fructose uptake remain poorly understood. Here, we demonstrate that MASLD livers exhibit increased uptake of fructose-derived carbons compared to healthy livers and identify that the MASLD hepatocyte secretome can increase fructose metabolism. By performing fractionation and untargeted proteomics, we uncover a role for Angiopoietin-like 3 (ANGPTL3) as a regulator of hepatic fructose metabolism, independent of its role as a lipoprotein lipase (LPL) inhibitor. Circulating ANGPTL3 levels increase in response to fructose exposure, consistent with an action as a fructose sensor. Angptl3 knockdown in the liver resulted in a significant reduction in the uptake of hepatic fructose metabolites in vivo and downregulation of the facilitative hepatic fructose transporter slc2a8 (GLUT8) and fructolysis enzymes. This work demonstrates the existence of extracellular control of hepatic fructose metabolism through ANGPTL3.

  View details for DOI 10.1016/j.celrep.2025.115962

  View details for PubMedID 40638391

• Genetic depletion of adipose-derived Isthmin-1 causes hepatic steatosis. Molecular metabolism Reghupaty, S. C., Coassolo, L., Zhao, M., Narasimhan, R. L., Patel, A., Lone, J., Bielczyk-Maczynska, E., Danneskiold-Samsoee, N. B., Brown, T., Jiang, Z., Li, V. L., Svensson, K. J. 2025: 102172

  Abstract

  Adipose tissue plays a critical role in obesity, as its dysfunction can impair lipid homeostasis and result in lipid overflow and ectopic lipid deposition in the liver. We previously demonstrated that Isthmin-1 (Ism1) regulates glucose uptake into the adipose tissue and suppresses hepatic steatosis, but the role of adipose-derived Ism1 is unknown. Here, we investigate the role of adipose-derived Ism1 in metabolic health and its impact on hepatic steatosis and lipid metabolism.In this study, we employed both a genetic knockout approach, selectively deleting Ism1 in adipose tissue of mice (AdipoQ-Ism1-KO), and a pharmacological approach by administering recombinant Ism1 protein to mice. These mice were subjected to a high fat-high fructose diet to simulate conditions that promote Metabolic-dysfunction Associated Steatotic Liver Disease (MASLD).AdipoQ-Ism1-KO are of normal weight, but prone to severe hepatic steatosis in response to high fat-high fructose feeding. Lipidomic profiling through untargeted analyses in both gain-of-function and loss-of-function models was used to assess changes in hepatic lipid homeostasis. These results provide in vivo genetic support for the role of Ism1 as a regulator of the adipose-hepatic axis.Collectively, these data demonstrate that loss of adipose-derived Ism1 disrupts lipid homeostasis and accelerates the development of hepatic steatosis. This study provides a genetic basis for Ism1's involvement in metabolic regulation, suggesting a potential therapeutic target for treating metabolic disorders.

  View details for DOI 10.1016/j.molmet.2025.102172

  View details for PubMedID 40436204

• Harnessing the power of peptides: advances and collaborations in biochemistry. Trends in biochemical sciences Coassolo, L., Svensson, K. J., Gare, C. L., White, A. M., Malins, L. R., Smith, A. B., Chekan, J. R. 2025

  View details for DOI 10.1016/j.tibs.2025.05.001

  View details for PubMedID 40410072

• Understanding peptide hormones: from precursor proteins to bioactive molecules. Trends in biochemical sciences Coassolo, L., Wiggenhorn, A., Svensson, K. J. 2025

  Abstract

  Peptide hormones are fundamental regulators of biological processes involved in homeostasis regulation and are often dysregulated in endocrine diseases. Despite their biological significance and established therapeutic potential, there is still a gap in our knowledge of their processing and post-translational modifications, as well as in the technologies for their discovery and detection. In this review, we cover insights into the peptidome landscape, including the proteolytic processing and post-translational modifications of peptide hormones. Understanding the full landscape of peptide hormones and their modifications could provide insights into leveraging proteolytic mechanisms to identify novel peptides with therapeutic potential. Therefore, we also discuss the need for future research aiming at better predicting, detecting, and characterizing new peptides with biological activities.

  View details for DOI 10.1016/j.tibs.2025.03.014

  View details for PubMedID 40234176

• Prohormone cleavage prediction uncovers a non-incretin anti-obesity peptide. Nature Coassolo, L., B Danneskiold-Samsøe, N., Nguyen, Q., Wiggenhorn, A., Zhao, M., Wang, D. C., Toomer, D., Lone, J., Wei, Y., Patel, A., Liparulo, I., Kavi, D., Wat, L. W., Reghupaty, S. C., Kim, J. J., Asemi, T., Bielczyk-Maczynska, E., Li, V. L., Moya-Garzon, M. D., Krentz, N. A., Stahl, A., Chou, D. H., Luo, L., Svensson, K. J. 2025

  Abstract

  Peptide hormones, a class of pharmacologically active molecules, have a critical role in regulating energy homeostasis. Prohormone convertase 1/3 (also known as PCSK1/3) represents a key enzymatic mechanism in peptide processing, as exemplified with the therapeutic target glucagon-like peptide 1 (GLP-1)1,2. However, the full spectrum of peptides generated by PCSK1 and their functional roles remain largely unknown. Here we use computational drug discovery to systematically map more than 2,600 previously uncharacterized human proteolytic peptide fragments cleaved by prohormone convertases, enabling the identification of novel bioactive peptides. Using this approach, we identified a 12-mer peptide, BRINP2-related peptide (BRP). When administered pharmacologically, BRP reduces food intake and exhibits anti-obesity effects in mice and pigs without inducing nausea or aversion. Mechanistically, BRP administration triggers central FOS activation and acts independently of leptin, GLP-1 receptor and melanocortin 4 receptor. Together, these data introduce a method to identify new bioactive peptides and establish pharmacologically that BRP may be useful for therapeutic modulation of body weight.

  View details for DOI 10.1038/s41586-025-08683-y

  View details for PubMedID 40044869

  View details for PubMedCentralID 4838524

• A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites. Cell Moya-Garzon, M. D., Wang, M., Li, V. L., Lyu, X., Wei, W., Tung, A. S., Raun, S. H., Zhao, M., Coassolo, L., Islam, H., Oliveira, B., Dai, Y., Spaas, J., Delgado-Gonzalez, A., Donoso, K., Alvarez-Buylla, A., Franco-Montalban, F., Letian, A., Ward, C. P., Liu, L., Svensson, K. J., Goldberg, E. L., Gardner, C. D., Little, J. P., Banik, S. M., Xu, Y., Long, J. Z. 2024

  Abstract

  β-Hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve the interconversion of BHB and primary energy intermediates. Here, we identify a previously undescribed BHB secondary metabolic pathway via CNDP2-dependent enzymatic conjugation of BHB and free amino acids. This BHB shunt pathway generates a family of anti-obesity ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. The most abundant BHB-amino acid, BHB-Phe, is a ketosis-inducible congener of Lac-Phe that activates hypothalamic and brainstem neurons and suppresses feeding. Conversely, CNDP2-KO mice exhibit increased food intake and body weight following exogenous ketone ester supplementation or a ketogenic diet. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, enzymatic amino acid BHB-ylation defines a ketone shunt pathway and bioactive ketone metabolites linked to energy balance.

  View details for DOI 10.1016/j.cell.2024.10.032

  View details for PubMedID 39536746

• AlphaFold2 enables accurate deorphanization of ligands to single-pass receptors. Cell systems Banhos Danneskiold-Samsoe, N., Kavi, D., Jude, K. M., Nissen, S. B., Wat, L. W., Coassolo, L., Zhao, M., Santana-Oikawa, G. A., Broido, B. B., Garcia, K. C., Svensson, K. J. 2024

  Abstract

  Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold2 (AF2) as a screening approach to identify extracellular ligands to single-pass transmembrane receptors. Key to the approach is the optimization of AF2 input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. The predictions were performed on ligand-receptor pairs not used for AF2 training. We demonstrate high discriminatory power and a success rate of close to 90% for known ligand-receptor pairs and 50% for a diverse set of experimentally validated interactions. Further, we show that screen accuracy does not correlate linearly with prediction of ligand-receptor interaction. These results demonstrate a proof of concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

  View details for DOI 10.1016/j.cels.2024.10.004

  View details for PubMedID 39541981

• A secondary β-hydroxybutyrate metabolic pathway linked to energy balance. bioRxiv : the preprint server for biology Moya-Garzon, M. D., Wang, M., Li, V. L., Lyu, X., Wei, W., Tung, A. S., Raun, S. H., Zhao, M., Coassolo, L., Islam, H., Oliveira, B., Dai, Y., Spaas, J., Delgado-Gonzalez, A., Donoso, K., Alvarez-Buylla, A., Franco-Montalban, F., Letian, A., Ward, C., Liu, L., Svensson, K. J., Goldberg, E. L., Gardner, C. D., Little, J. P., Banik, S. M., Xu, Y., Long, J. Z. 2024

  Abstract

  β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.

  View details for DOI 10.1101/2024.09.09.612087

  View details for PubMedID 39314488

  View details for PubMedCentralID PMC11418978

• PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity. Nature Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Li, L., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

  View details for DOI 10.1038/s41586-024-07801-6

  View details for PubMedID 39112712

  View details for PubMedCentralID 3501277

• A class of secreted mammalian peptides with potential to expand cell-cell communication. Nature communications Wiggenhorn, A. L., Abuzaid, H. Z., Coassolo, L., Li, V. L., Tanzo, J. T., Wei, W., Lyu, X., Svensson, K. J., Long, J. Z. 2023; 14 (1): 8125

  Abstract

  Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

  View details for DOI 10.1038/s41467-023-43857-0

  View details for PubMedID 38065934

  View details for PubMedCentralID 5946320

• Rapid and accurate deorphanization of ligand-receptor pairs using AlphaFold. bioRxiv : the preprint server for biology Danneskiold-Samsøe, N. B., Kavi, D., Jude, K. M., Nissen, S. B., Wat, L. W., Coassolo, L., Zhao, M., Santana-Oikawa, G. A., Broido, B. B., Garcia, K. C., Svensson, K. J. 2023

  Abstract

  Secreted proteins are extracellular ligands that play key roles in paracrine and endocrine signaling, classically by binding cell surface receptors. Experimental assays to identify new extracellular ligand-receptor interactions are challenging, which has hampered the rate of novel ligand discovery. Here, using AlphaFold-multimer, we developed and applied an approach for extracellular ligand-binding prediction to a structural library of 1,108 single-pass transmembrane receptors. We demonstrate high discriminatory power and a success rate of close to 90 % for known ligand-receptor pairs where no a priori structural information is required. Importantly, the prediction was performed on de novo ligand-receptor pairs not used for AlphaFold training and validated against experimental structures. These results demonstrate proof-of-concept of a rapid and accurate computational resource to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

  View details for DOI 10.1101/2023.03.16.531341

  View details for PubMedID 36993313

  View details for PubMedCentralID PMC10055078

• Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution. iScience Coassolo, L., Liu, T., Jung, Y., Taylor, N. P., Zhao, M., Charville, G. W., Nissen, S. B., Yki-Jarvinen, H., Altman, R. B., Svensson, K. J. 2023; 26 (1): 105802

  Abstract

  Non-alcoholic fatty liver disease is a heterogeneous disease with unclear underlying molecular mechanisms. Here, we perform single-cell RNA sequencing of hepatocytes and hepatic non-parenchymal cells to map the lipid signatures in mice with non-alcoholic fatty liver disease (NAFLD). We uncover previously unidentified clusters of hepatocytes characterized by either high or low srebp1 expression. Surprisingly, the canonical lipid synthesis driver Srebp1 is not predictive of hepatic lipid accumulation, suggestive of other drivers of lipid metabolism. By combining transcriptional data at single-cell resolution with computational network analyses, we find that NAFLD is associated with high constitutive androstane receptor (CAR) expression. Mechanistically, CAR interacts with four functional modules: cholesterol homeostasis, bile acid metabolism, fatty acid metabolism, and estrogen response. Nuclear expression of CAR positively correlates with steatohepatitis in human livers. These findings demonstrate significant cellular differences in lipid signatures and identify functional networks linked to hepatic steatosis in mice and humans.

  View details for DOI 10.1016/j.isci.2022.105802

  View details for PubMedID 36636354

  View details for PubMedCentralID PMC9830221

• STARD3: A New Biomarker in HER2-Positive Breast Cancer. Cancers Lodi, M., Voilquin, L., Alpy, F., Moliere, S., Reix, N., Mathelin, C., Chenard, M., Tomasetto, C. 2023; 15 (2)

  Abstract

  Pathological complete response (pCR) after neoadjuvant systemic treatment (NST) is an important prognostic factor in HER2-positive breast cancer. The majority of HER2-positive breast cancers are amplified at the HER2 gene locus, several genes are co-amplified with HER2, and a subset of them are co-expressed. The STARD3 gene belongs to the HER2 amplicon, and its role as a predictive marker was never addressed. The objective of this study was to investigate the predictive value of STARD3 protein expression on NST pathological response in HER2-positive breast cancer. In addition, we studied the prognostic value of this marker.METHODS: We conducted a retrospective study between 2007 and 2020 on 112 patients with non-metastatic HER2-positive breast cancer treated by NST and then by surgery. We developed an immunohistochemistry assay for STARD3 expression and subcellular localization and determined a score for STARD3-positivity. As STARD3 is an endosomal protein, its expression was considered positive if the intracellular signal pattern was granular.RESULTS: In this series, pCR was achieved in half of the patients. STARD3 was positive in 86.6% of cases and was significantly associated with pCR in univariate analysis (p = 0.013) and after adjustment on other known pathological parameters (p = 0.044). Performances on pCR prediction showed high sensitivity (96%) and negative predictive value (87%), while specificity was 23% and positive predictive value was 56%. Overall, specific, relapse-free, and distant metastasis-free survivals were similar among STARD3 positive and negative groups, independently of other prognosis factors.CONCLUSION: NST is an opportunity for HER2-positive cancers. In this series of over a hundred HER2-positive and non-metastatic patients, a STARD3-negative score was associated with the absence of pathological complete response. This study suggests that determining STARD3 overexpression status on initial biopsies of HER2-positive tumors is an added value for the management of a subset of patients with high probability of no pathological response.

  View details for DOI 10.3390/cancers15020362

  View details for PubMedID 36672312

• New players of the adipose secretome: Therapeutic opportunities and challenges. Current opinion in pharmacology Coassolo, L., Dannieskiold-Samsøe, N. B., Zhao, M., Allen, H., Svensson, K. J. 2022; 67: 102302

  Abstract

  Adipose tissue is a functional endocrine organ comprised of adipocytes and other cell types that are known to secrete a multiplicity of adipose-derived factors, including lipids and proteins. It is well established that adipose tissue and its secretome can impact systemic energy homeostasis. The endocrine and paracrine effects of adipose-derived factors have been widely studied over the last several decades. Owing to technological advances in genomics and proteomics, several additional adipose-derived protein factors have recently been identified. By learning from previous efforts, the next challenge will be to leverage these discoveries for the prevention or treatment of metabolic disorders. Here, we discuss recently discovered adipose-derived proteins secreted from white or brown adipose tissue and the opportunities and challenges of translating these biological findings into disease therapeutics.

  View details for DOI 10.1016/j.coph.2022.102302

  View details for PubMedID 36195010

• Phosphoproteomic mapping reveals distinct signaling actions and activation of muscle protein synthesis by Isthmin-1 eLife Zhao, M., Banhos Danneskiold-Samsøe, N., Ulicna, L., Nguyen, Q., Voilquin, L., Lee, D. E., White, J. P., Jiang, Z., Cuthbert, N., Paramasivam, S., Bielczyk-Maczynska, E., van Rechem, C., Svensson, K. J. 2022

  View details for DOI 10.7554/eLife.80014

• Isthmin-1 is an adipokine that promotes glucose uptake and improves glucose tolerance and hepatic steatosis. Cell metabolism Jiang, Z., Zhao, M., Voilquin, L., Jung, Y., Aikio, M. A., Sahai, T., Dou, F. Y., Roche, A. M., Carcamo-Orive, I., Knowles, J. W., Wabitsch, M., Appel, E. A., Maikawa, C. L., Camporez, J. P., Shulman, G. I., Tsai, L., Rosen, E. D., Gardner, C. D., Spiegelman, B. M., Svensson, K. J. 2021

  Abstract

  With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

  View details for DOI 10.1016/j.cmet.2021.07.010

  View details for PubMedID 34348115

• FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts EMBO JOURNAL Di Mattia, T., Martinet, A., Ikhlef, S., McEwen, A. G., Nomine, Y., Wendling, C., Poussin-Courmontagne, P., Voilquin, L., Eberling, P., Ruffenach, F., Cavarelli, J., Slee, J., Levine, T. P., Drin, G., Tomasetto, C., Alpy, F. 2020; 39 (23): e104369

  Abstract

  Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

  View details for DOI 10.15252/embj.2019104369

  View details for Web of Science ID 000585124000001

  View details for PubMedID 33124732

  View details for PubMedCentralID PMC7705450

• Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides BIOCONJUGATE CHEMISTRY Stoessel, A., Groysbeck, N., Guyot, L., Barret, L., Nomine, Y., Nguekeu-Zebaze, L., Bender, A., Voilquin, L., Lutz, T., Pallaoro, N., Blocat, M., Deville, C., Masson, M., Zuber, G., Chatton, B., Donzeau, M. 2020; 31 (10): 2421-2430

  Abstract

  Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

  View details for DOI 10.1021/acs.bioconjchem.0c00482

  View details for Web of Science ID 000584490900017

  View details for PubMedID 32996763

• Another hijack! Some enteroviruses co-opt the c10orf76/PI4KB complex for their own good. EMBO reports Voilquin, L., Di Mattia, T., Alpy, F. 2020; 21 (2): e49876

  Abstract

  Enteroviruses, members of the Picornaviridae family, are non-enveloped and single-stranded RNA viruses responsible for several human diseases. During infection, these viruses build membrane-bound organelles, called replication organelles (ROs), where new virions are assembled. ROs are highly enriched in phosphatidylinositol 4-phosphate (PI4P) produced by the host lipid kinase PI4KB. In this issue of EMBO Reports, McPhail et al [1] characterize a complex, formed by PI4KB and the c10orf76 protein, which is involved in PI4P production. They show that this machinery is hijacked by specific enteroviruses such as coxsackievirus A10 for their replication.

  View details for DOI 10.15252/embr.201949876

  View details for PubMedID 31919962

  View details for PubMedCentralID PMC7001151

• STARD3: A Swiss Army Knife for Intracellular Cholesterol Transport STARD3: A Swiss Army Knife for Intracellular Cholesterol Transport Voilquin, L., Lodi, M., Di Mattia, T., Chenard, M., Mathelin, C., Alpy, F., Tomasetto, C. 2019
• Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4 INTRACELLULAR LIPID TRANSPORT: METHODS AND PROTOCOLS Wilhelm, L. P., Voilquin, L., Kobayashi, T., Tomasetto, C., Alpy, F. edited by Drin, G. 2019; 1949: 137-152

  Abstract

  Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-β-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.

  View details for DOI 10.1007/978-1-4939-9136-5_11

  View details for Web of Science ID 000608835800012

  View details for PubMedID 30790254