bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.007286; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The type 2 diabetes gene product STARD10 is a phosphoinositide binding protein that controls insulin secretory granule biogenesis Gaelle R. Carrat1, Elizabeth Haythorne1, Alejandra Tomas1, Leena Haataja2, Andreas Müller3,4,5,6, Peter Arvan2, Alexandra Piunti1,7, Kaiying Cheng8, Mutian Huang1, Timothy J. Pullen1,9, Eleni Georgiadou1, Theodoros Stylianides10, Nur Shabrina Amirruddin11,12, Victoria Salem 1,13, Walter Distaso14, Andrew Cakebread 15, Kate J.Heesom16, Philip A. Lewis16, David J. Hodson17, Linford J. Briant18, Annie C.H. Fung19, Richard B. Sessions20, Fabien Alpy21, Alice P.S. Kong19, Peter I. Benke22, Federico Torta22, Adrian Kee Keong Teo11,12, 23, Isabelle Leclerc1, Michele Solimena 3,4,5,6 , Dale B. Wigley8 and Guy A. Rutter1* 1 - Section of Cell Biology and Functional Genomics, Imperial College London, du Cane Road, London W12 0NN, UK. 2 - Division of Metabolism, Endocrinology & Diabetes, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA. 3 - Molecular Diabetology, University Hospital and Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany. 4 - Paul Langerhans Institute Dresden (PLID) of the Helmholtz Center Munich, University Hospital Carl Gustav Carus and Faculty of Medicine of the TU Dresden, Dresden, Germany. 5 - German Center for Diabetes Research (DZD e.V.), Neuherberg, Germany. 6 - Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany. 7 - Lille 1 University - Science and Technology, Cité Scientifique 59655 Villeneuve d'Ascq Cedex, France. 8 - Section of Structural Biology, Department of Medicine, Imperial College London, London, United Kingdom. 9 - Department of Diabetes, Faculty of Life Science and Medicine, King’s College London, London, UK 10 - Loughborough University, Centre of Innovative and Collaborative Construction Engineering, Leicestershire, LE11 3TU, UK. 11 - Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), A*STAR, Proteos, Singapore 138673, Singapore 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.007286; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 12 - Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore 13 - Section of Investigative Medicine, Department of Medicine, Imperial College London, du Cane Road, London W12 0NN, UK. 14 - Imperial College Business School, Imperial College London, Exhibition Road, London SW7 2AZ 15 - London Metallomics Facility, King’s College London, Strand, London WC2R 2LS 16 - Proteomics Facility, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol, UK. 17 - Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, U.K., Institute of Metabolism and Systems Research, University of Birmingham, Edgbaston, U.K., Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, Midlands, UK. 18 - Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LE, UK. 19 - Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong. 20 - School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK. 21 - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) ; Institut National de la Santé et de la Recherche Médicale (INSERM) U1258; Centre National de la Recherche Scientifique (CNRS) UMR 7104; Université de Strasbourg ;1 rue Laurent Fries, 67404 Illkirch, France. 22 - Singapore Lipidomics Incubator, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore; 8 Medical Drive, Singapore 117596, Singapore 23 - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, Singapore *To whom correspondence should be addressed at [email protected]; +44 (0)20 7594 3340 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.007286; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Objective Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids, and thus the pathways through which STARD10 regulates β- cell function, are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and its effect on proinsulin processing and insulin granule biogenesis and maturation. Methods We used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStarD10KO) and performed electron microscopy, pulse-chase, RNA sequencing and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay were performed on purified STARD10 protein. Results βStarD10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of “rod-like” dense cores. Correspondingly, basal secretion of proinsulin was increased. Amongst the differentially expressed genes in βStarD10KO islets, expression of the phosphoinositide binding proteins Pirt and Synaptotagmin 1 were decreased while lipidomic analysis demonstrated changes in phosphatidyl inositol levels. The inositol lipid kinase PIP4K2C was also identified as a STARD10 binding partner. STARD10 bound to inositides 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.007286; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. phosphorylated at the 3’ position and solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides. Conclusion Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis and insulin processing. Keywords: Type 2 diabetes; pancreatic β-cell; lipid transporter; insulin granule biogenesis; phosphoinositides Abbreviations: BUDE: Bristol University Docking Engine; GFP: Green Fluorescent Protein; + GWAS : Genome Wide Association Study; KATP: ATP-sensitive K channels; HRP: Horseradish Peroxidase; Kir6.2: Kcnj11: Potassium Inwardly Rectifying Channel Subfamily J Member 11; KOMP: NIH Knock-Out Mouse Project; ICP/MS: Inductively coupled plasma mass spectrometry; IMPC: International Mouse Phenotyping Consortium; LPC: Lysophosphatidylcholine; MBP: Maltose Binding Protein; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PI: Phosphatidylinositol; PI(3)P: Phosphatidylinositol 3-phosphate; PI(4,5)P2: PIP2, Phosphatidylinositol 4,5-bisphosphate; PI(5)P: Phosphatidylinositol 5- phosphate; PIP: phosphatidylinositol phosphate, phosphatidylinositide; Pip4k2c: phosphatidylinositol 5-phosphate 4-kinase type-2 gamma; Pirt: phosphoinositide-interacting regulator of transient receptor potential channels; RNAseq: RNA sequencing; Ptbp1: Polypyrimidine tract-binding protein 1; Slc30a8: ZnT8, Solute Carrier Family 30 Member 8; SNARE: Soluble N-ethylmaleimide sensitive factor Attachment protein Receptor; STARD10: StAR Related Lipid Transfer Domain Containing 10; Syt1: Synaptotagmin 1; Sytl4: synaptotagmin-like 4, granuphilin; TMT: Tandem Mass Tag; TRP: Transient receptor potential. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.25.007286; this version posted March 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1. Introduction Diabetes mellitus is characterised by high blood glucose and currently affects around 8.5% of the population worldwide [1]. Normal glucose homeostasis requires the processing of proinsulin and the storage of the mature hormone within dense core granules in the pancreatic β-cell [2]. Glucose-induced insulin secretion involves glucose uptake and metabolism through the glycolytic pathway, increased ATP production by the mitochondria and the closure of ATP- + sensitive K channels (KATP). Subsequent depolarization of the plasma membrane leads to opening of voltage-gated Ca2+ channels, Ca2+ dependent assembly