Functional Studies of VANGL Mutations Associated with Neural Tube Defects
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Functional studies of VANGL mutations associated with neural tube defects. Alexandra Iliescu Department of Biochemistry McGill University Montreal, Quebec, Canada February 2015 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy. © Alexandra Iliescu, 2015 1 Abstract Loop-tail (Lp) mice show a very severe neural tube defect (craniorachischisis) caused by mutations in the Vangl2 gene (D255E, R259L, S464N). Mammalian VANGL1 and VANGLl2 are membrane proteins that play critical roles in development such as establishing planar cell polarity (PCP) in epithelial layers and convergent extension (CE) movements during neural tube closure. In this thesis, the molecular mechanism of VANGL proteins was investigated. Chapters 2 and 6 explore structure-function relationships, while Chapters 3, 4 and 5 study the molecular basis of loss-of-function of mutations either Lp-associated or identified in human cases of neural tube defects (NTDs). In Chapter 2, we used epitope tagging and immunofluorescence to establish the secondary structure of VANGL proteins, including the number, position, and polarity of transmembrane domains. These studies indicate that VANGL proteins have a four-transmembrane domain structure and that both the amino and large carboxy termini portions of the protein are located intracellularly. Work performed in Chapters 3 and 4 show that all three Lp-associated mutations (D255E, R259L, D464N) share a common loss-of-function mechanism. While WT VANGL proteins are expressed at the plasma membrane of transfected MDCK cells, their site for biological function, Lp-associated mutations are retained intracellularly in the endoplasmic reticulum, have reduced half-life and are rapidly degraded in a proteasome-dependent fashion. These studies provided a biochemical framework for the study of recently identified mutations in VANGL1 and VANGL2 in sporadic or familial cases of NTDs. Chapter 5 focuses on two arginine residues R181 and R274, that are highly conserved in VANGL protein relatives, and that are found independently mutated in VANGL1 (R181Q and R274Q) and VANGL2 (R177H and R270H) in human cases of NTDs. Compared to WT, the R181Q and R274Q mutations also displayed impaired targeting to the plasma membrane, showed impaired stability and a large reduction in half-life. In Chapter 6 a set of linear deletions in the amino and carboxyl termini of VANGL1 were created to delineate the sequence determinants that are required for targeting these proteins to the plasma membrane. This work identified Vangl plasma membrane motifs to be restricted to the carboxy terminus, including the PDZ binding motif. 2 Résumé La souris loop-tail (Lp) meurt d’une anomalie très grave du tube neural (craniorachischisis) causée par des mutations dans le gène Vangl2 (D255E, R259L, S464N). Chez les mammifères, il existe deux gènes; Vangl1 et Vangl2 qui codent pour des protéines membranaires. Celles-ci jouent un rôle critique dans le développement, contrôlant l'établissement de la polarité cellulaire planaire (PCP) chez les cellules épithéliales ainsi que le processus d’extension convergente lors de la fermeture du tube neural. Dans cette thèse, le mécanisme moléculaire des protéines VANGL a été étudié. Les chapitres 2 et 6 enquêtent sur les relations entre la structure et la fonction de la protéine, tandis que les chapitres 3, 4 et 5 étudient le mécanisme moléculaire associé à la perte de fonction chez les mutations Lp ou bien chez celles identifiées chez des patients souffrant d’anomalies de tubes neuraux (ATN). Au chapitre 2, nous avons établi la structure secondaire des protéines VANGL, y compris le nombre, la position et la polarité des domaines transmembranaires. Ces études indiquent que ces protéines possèdent quatre domaines transmembranaires et que les deux extrémités amino et carboxy terminales sont situées au niveau intracellulaire. Le travail effectué dans les chapitres 3 et 4 montre que les trois mutations Lp (D255E, R259L, D464N) partagent un mécanisme de perte de fonction commun. Tandis que les protéines VANGL WT sont exprimées à la membrane cellulaire des cellules MDCK transfectées, les mutations Lp sont retenues dans le réticulum endoplasmique, elles possèdent une demi-vie réduite et sont rapidement dégradées par le protéasome. Ces études ont fourni un cadre de tests biochimiques pour analyser les mutations récemment trouvées dans VANGL1 et VANGL2 dans des cas sporadiques ou familiaux d’anomalies de tube neural. Le chapitre 5 se concentre sur deux arginines; R181 et R274, qui sont hautement conservées chez les homologues des protéines VANGL, et qui sont aussi mutées de façon indépendante dans VANGL1 (R181Q et R274Q) et VANGLl2 (R177H et R270H) dans des cas humains d’ATN. Comparativement au WT, les mutations R181Q et R274Q ne sont pas ciblées à la membrane plasmique, elles affichent une stabilité moindre ainsi qu’une réduction importante de la demi-vie. Dans le chapitre 6, une série de délétions linéaires à l'extrémité amino-et carboxy- terminales de VANGL1 ont été créés afin de déterminer les signaux dans la séquence nécessaires au ciblage de ces protéines à la membrane cellulaire. Ces travaux ont identifié que ces motifs se situent à l'extrémité carboxy-terminale, y compris au domaine de liaison PDZ. 3 Preface The work described in Chapters 2, 3, 4, 5 and 6 of this thesis are published as follows: Chapter 2: Iliescu A, Gravel M, Horth C, Apuzzo S, Gros P. (2011) Transmembrane topology of mammalian planar cell polarity protein VANGL1. Biochemistry. 50(12):2274-82. Chapter 3: Gravel M, Iliescu A, Horth C, Apuzzo S, Gros P. (2010) Molecular and cellular mechanisms underlying neural tube defects in the loop-tail mutant mouse. Biochemistry. 49(16):3445-55. Chapter 4: Iliescu A, Gravel M, Horth C, Kibar Z, Gros P. (2011) Loss of membrane targeting of VANGL proteins causes neural tube defects. Biochemistry. 50(5):795-804. Chapter 5: Iliescu A, Gravel M, Horth C, Gros P. (2014) Independent mutations at Arg181 and Arg274 of VANGL proteins that are associated with neural tube defects in humans decrease protein stability and impair membrane targeting. Biochemistry. (Epub ahead of print) Chapter 6: Iliescu A, Gros P. (2014) The intracellular carboxy terminal domain of VANGL proteins contains plasma membrane targeting elements. Protein Science. 23(4):337-43. 4 Contribution of Authors Chapter 2: The work described in this chapter was mainly performed by myself. Prior to my arrival in the lab, Sergio Appuzo created all six c-Myc/HA-tagged VANGL1 constructs used in this study. In order to facilitate the detection of positively transfected clones, Dr. Michel Gravel fused the constructs in-frame to GFP. I performed hydrophathy profiling, stably transfected and expressed the constructs in MDCK cells, characterized the subcellular localization of the recombinant proteins and quantified cell surface expression. Cynthia Horth provided valuable microscopy advice and helped perform some of the image acquisition. All the figures in this manuscript are based on experiments that I performed. I constructed all the figures and wrote the first draft of the manuscript. Chapter 3: The work described in this chapter was produced in collaboration with Dr. Michel Gravel and Cynthia Horth. I performed the subcellular localization and membrane targeting studies. I participated, along with Dr. Gravel in constructing the figures and the writing of the manuscript. Dr. Elena Torban did a critical reading of the manuscript. Dr. Gravel helped in the creation of the mutant constructs. Chapter 4: The work described in this chapter was mainly performed by myself and in collaboration with Dr. Michel Gravel and Cynthia Horth. Dr. Gravel helped in the creation of the mutant constructs. I performed the subcellular localization and membrane targeting studies. Dr. Michel Gravel carried out the Pulse-Chase experiments (figure 6) and Cynthia Horth performed the coexpression of WT and Lp mutations in MDCK cells (figure 9). I constructed all the figures and wrote the first draft of the manuscript. Dr. Zoha Kibar and Dr. Elena Torban did a critical reading of the manuscript. Chapter 5: The work described in this chapter was mainly performed by myself. Prior to my arrival on the project, Dr. Zoha Kibar initially sequenced the cohort of human patients and identified all three 5 mutations (R181Q, L202F and R274Q). I created the mutants in collaboration with Dr. Gravel and I expressed the constructs, characterized their proper membrane targeting, subcellular localization, protein stability, proteasomal dependent degradation and determined the half-life of the proteins. Michel Gravel performed the pulse-chase of the R181Q mutant and Cynthia Horth helped select positive clones and perform some of the immunofluorescence and image acquisition. I constructed all the figures and wrote the first draft of the manuscript. Chapter 6: The work described in this chapter is essentially my own. Dr. Michel Gravel provided valuable technical advice on the strategy for creating the deletion constructs and Jean-Daniel Castonguay, an undergraduate student that I supervised, helped me with the molecular cloning steps. I created all the deletion constructs, stably expressed them in MDCK cells and characterized their subcellular localization. All the figures in this manuscript are based on experiments that I performed. I constructed all the figures and wrote the first draft of the manuscript. Dr. Michel Gravel did a critical reading of the manuscript.