The effect of the polyglutamine expansion of the Androgen Receptor on the ubiquitin proteasome system for protein degradation Thomas Carr Scanlon Department of Human Genetics McGill University, Montreal, Quebec August, 2007 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Thomas Scanlon, 2007 i Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-53327-7 Our file Notre reference ISBN: 978-0-494-53327-7 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Nnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada ABSTRACT The Androgen Receptor (AR; Xql 1.2-ql2) is a well-characterized X-linked gene. The AR protein functions primarily as a steroid-activated transcription factor. A structural feature of the AR transactivational domain (TAD) called the polyglutamine tract will be the primary focus of this thesis. The polyglutamine tract is an uninterrupted stretch of glutamine residues, polymorphic in length, that contributes to the transactivational potential of the receptor, and has also been demonstrated to be the causative agent in Spinal and Bulbar Muscular Atrophy (SBMA). SBMA is a late-onset neurological disease caused by an expansion of the polyglutamine tract in the AR. Immunohistopathological investigations of affected tissue in SBMA patients reveal strong anti-AR staining in discrete, electrodense regions, termed "inclusion bodies" or "protein aggregates". This finding has been corroborated by various transfection studies comparing wild type and polyglutamine tract-expanded AR, leading to a large variety of hypotheses regarding the formation and potential toxicity of .these structures. The consistent observation of accumulated, insoluble polyglutamine - expanded protein suggests a malfunction of the normal mechanisms of protein clearance. The primary goal of this thesis is to explore the effect of the polyglutamine expansion mutation in the AR on the ubiquitin proteasome system of protein degradation (UPS). To this end, several strategies have been implemented. A classic proteasome reporter cell line was used to create a cell culture model of SBMA to show evidence for involvement of the UPS in SBMA. To investigate the putative nuclear dependence of polyglutamine-mediated toxicity, transfection studies using a mutant AR with defective n nuclear localization signal were also performed in this cell line. In an attempt to unequivocally demonstrate polyglutamine-expanded protein degradation by the proteasome, efforts were made to create an entirely in vitro method to test AR degradation by the UPS. Human proteasomes were successfully purified by a novel affinity chromatography method towards this end. Finally, the AR TAD with various polyglutamine tract lengths was bacterially- expressed and highly purified for structural analysis of the polyglutamine tract in a native protein. Dynamic light scattering experiments demonstrated expanded polyQ tracts exhibit biochemical aggregative properties. Circular dichroism spectra were collected for each polyQ tract variant. Results indicated any length polyQ tract is an strong structure- breaking sequence. Furthermore, the expansion mutant exhibited pronounced P-sheet strucutre absent from all other variants. In sum, structural investigations suggest a molten globule structure for the wild type AR-TAD. in RESUME Le recepteur androgene (AR), un gene situe sur le chromosome X en position Xqll.2-ql2 est, a prime abord, un facteur transcriptionnel active via les hormones steroidiennes. Le sujet de cette these se concentre suf un domaine structural du recepteur androgene, lequel s'avere necessaire pour l'activite transcriptionnelle du recepteur. Ce domaine nomme «polyglutamine tract» consiste en une sequence repetitive et ininterrompue de glutamine dont le nombre est polymorphe. Le nombre de repetition affecte directement le potentiel transactivationnel du recepteur et s'avere etre l'element causal de l'atrophie spinobulbaire (SBMA; SpinoBulbar Muscular Atrophy). L'atrophie spinobulbaire est une maladie neurologique se developpant tardivement chez l'adulte. Celle-ci est causee par une expansion du nombre de repetition de glutamine formant le « polyglutamine tract » du recepteur androgene. L'investigation par immunohistopathologie de tissus provenant de patients atteints de SBMA revele une concentration dense du recepteur androgene sous forme d'agregats proteiques nomme corps d'inclusion. Cette decouverte sur le potentiel toxique de ces structures a ete corroboree par plusieurs etudes de subexpression du gene AR normal (WT; Wild Type) ainsi que la version ayant subie une expansion du nombre de glutamine. L'observation constante des agregats insolubles de la proteine AR portant l'expansion de glutamine suggere un probleme au niveau de sa gestion par les mecanismes de degradation proteiques normaux. IV Le but premier de cette these est d'explorer les effets causes sur la degradation par le systeme ubiquitine/proteasome (UPS) du recepteur androgene a la suite d'une expansion du nombre de repetition de glutamine dans le gene. Pour ce faire, plusieurs strategies ont dues etre utilisees a l'aide d'une lignee cellulaire servant de modele pour l'atrophie spinobulbaire (SBMA). Pour montrer l'effet de la localisation nucleaire de la proteine du recepteur androgene portant une expansion de glutamine sur la toxicite cellulaire, des etudes de transfection de la proteine mutante portant aussi un signal de localisation nucleaire deficient ont ete utilisees. Afin de demontrer l'effet de l'expansion de la repetition de glutamine, des proteasomes humains ont ete purifies par chromatographic d'affinite afin de tester l'efficacite de la degradation de constructions du recepteur androgene arborant differentes longueurs de repetition de glutamine. v ACKNOWLEDGEMENTS In the spirit of full disclosure, it is acknowledged that the author was the beneficiary of excellent guidance and spirited discussion of this thesis and all aspects of science (ranging from the appropritately relevant to the wildly speculative) with my supervisor, Dr. Mark Trifiro. In addition, considerable time and effort in the design of experiments, interpretation of results and suggestion of controls was provided by several members of the Trifiro laboratory: Dr. Lenore K. Beitel, Carlos Alvarado, Rose Lumbroso, and Dr. Bruce Gottlieb. It should be expressly stated that tandem mass spectrometry of purified proteasome fractions was performed on a fee-for-service basis by Dr. Marcos DiFalco at the McGill University and Genome Quebec Innovation Centre. Database mining of the resultant LC-MS/MS data was performed by the author with the assistance of Dr. Beniot Houle and Yannic Richard of the same institution. Finally, the LC-MS/MS data from proteasome purifications was searched against a randomized version of the NCBI database by Dr. Rob Kearney, also of the McGill University and Genome Quebec Innovation Centre. Materials were gathered from various sources for completion of the research described in this thesis. Dr. Ron Kopito provided the HEK-293 GFP" cell line. In addition, several expression vectors for transfection experiments had already been constructed by various members of the Trifiro laboratory including EBFP-AR -Q20 and - Q50; ARANLS -Q20 and -Q50 plasmids, and the bacterial N-terminal TAD expression vectors. In addition, the GST-UBL was provided by Dr. Juli Feigon, University of California at Los Angeles, and the pMT123 expression vector was a gift from Dr. Simon Wing, McGill University. All other DNA vectors were constructed by the author. Finally, it would have been impossible to complete this work without the unconditional love and suppport from my parents, Timothy and Barbara Scanlon. Additionally, I would like to express gratitude for which there are no words to