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An Investigation of the Role of Creb Deubiquitinating Enzyme in the Regulation of Carbon Metabolism in Aspergillus Nidulans

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An Investigation of the Role of Creb Deubiquitinating Enzyme in the Regulation of Carbon Metabolism in Aspergillus Nidulans An investigation of the role of CreB deubiquitinating enzyme in the regulation of carbon metabolism in Aspergillus nidulans By Md Ashiqul Alam M.Pharm (Pharmaceutical Chemistry) Submitted in total fulfilment of the requirements of the Degree of Doctor of Philosophy Department of Genetics & Evolution School Biological Sciences Faculty of Sciences The University of Adelaide September, 2016 Table of Contents Thesis Abstract i Declaration iii Acknowledgement iv List of Publications v Chapter 1: Introduction and Literature Review 1.1 Introduction 1 1.2 Carbon Catabolite Repression 2 1.3 Carbon catabolite repression in Saccharomyces cerevisiae 2 1.3.1 Mig1P & other repressor 3 1.3.2 Snf1p 7 1.3.3 Snf4p 9 1.3.4 Reg1p & Glc7p 9 1.3.5 Gal83p, Sip1p & Sip2p 10 1.3.6 Ssn6p & Tup1p 10 1.3.7 Glucose Transporters 11 1.3.8 Snf3p & Rgt2p 12 1.3.9 Hxk2p 13 1.3.10 Rgt1p 15 1.4 Carbon catabolite repression in Aspergillus nidulans 16 1.4.1 CreA 16 1.4.2 CreB & CreC 20 1.4.3 CreD 22 1.4.4 AcrB 23 1.4.5 Hexose transporters & signalling pathways 23 1.5 Ubiquitination & Deubiquitination system 26 1.6 Carbon catabolite repression in other filamentous fungi 29 1.7 Carbon catabolite repression: S. cerevisiae vs A. nidulans 32 1.8 Model of CCR mechanism in A. nidulans 33 1.9 Unresolved questions in CCR 34 1.10 A. nidulans as an experimental organism 35 1.11 Aims of the Study 38 Table of Contents Chapter 2: The CreB deubiquitinating enzyme does not directly target 41 the CreA repressor protein in Aspergillus nidulans Supplementary 62 Chapter 3: Proteins interacting with CreA and CreB in the carbon 68 catabolite repression network in Aspergillus nidulans Supplementary 83 Chapter 4: ChIP-seq assay of CreA 87 4.1 Introduction 87 4.2 Materials and Methods 89 4.3 Results 90 4.3.1 ChIP-seq assay of CreA 90 4.3.2 Genome-wide CreA binding patterns in repressing 91 and derepressing conditions 4.3.3 Genome-wide direct target of CreA 97 4.4 Discussion 99 Supplementary attached as soft copy in CD Chapter 5: General Discussion 102 References 112 Thesis Abstract In A. nidulans, carbon catabolite repression is regulated by the global repressor protein CreA which, in the presence of repressing carbon sources, represses those genes that are required to utilize less preferable carbon sources. Mutational analyses suggested that ubiquitination, mediated by CreD together with ubiquitin ligase HulA, and deubiquitination, mediated by the deubiquitinating enzyme CreB, are involved in the regulatory pathway in A. nidulans. However, the molecular mechanisms are still unknown. Previously, partial loss-of-function alleles of creA and creB indicated genetic interaction, and this was extended to analysis of complete loss-of-function alleles. Both morphological and phenotypic analyses of the double null mutant confirmed genetic interactions between the genes. Moreover, RT-qPCR and enzyme assays also validated genetic interactions as the double null mutant showed synergistic effects for transcript levels and enzyme activity. Co-purifications of CreA and CreB expressed from their native promoters were used to determine whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitinating enzyme, and no direct or indirect physical interactions were identified. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. These results open up new questions regarding the molecular mechanism of CreA action, and how the ubiquitinating pathway involving CreB interacts with this regulatory network. To identify any possible protein(s) that may form a bridge between CreA and CreB, independently purified lysates were analysed by mass spectrometry and, for both CreA and CreB, proteins were identified in repressing and derepressing conditions. Orthologues of the co-purified proteins were identified in S. cerevisiae and humans. Functional annotation analysis revealed that proteins that preferentially interact with CreA in repressing conditions include histones and the histone transcription regulator 3, Hir3. Proteins interacting with CreB were involved in cellular transportation and organization. Similar findings were obtained using yeast and human orthologues, although the yeast background generated a number of other biological processes involving Mig1p which were not present in the A. nidulans or human background Thesis Abstract Page i analyses. Hir3 was present in repressing conditions for CreA, and in both growth conditions for CreB, suggesting that Hir3, or proteins interacting with Hir3, could be a possible target of CreB. Earlier, genome-wide microarray analysis showed that CreA was involved in the transcriptional regulation of a significant number of genes in A. nidulans, however, this approach cannot show whether the targets are directly or indirectly regulated. To identify the direct targets, and whether CreA binds in derepressing conditions, ChIP-seq analyses were performed. CreA constitutively bound to the promoters of target genes in both growth conditions, indicating that the function of CreA may be controlled on the chromatin by post-translational modifications. A total of 1946 unique targets were identified for both strains in repressing and derepressing conditions including genes that are involved in carbohydrate metabolic/catabolic processes, alcohol biosynthetic processes, secondary metabolism, and sugar and amino acid transporters. Thesis Abstract Page ii Declaration I certify that this work contains no material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. In addition, I certify that no part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of the University of Adelaide and where applicable, any partner institution responsible for the joint-award of this degree. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. I acknowledge that copyright of published works contained within this thesis resides with the copyright holder(s) of those works. I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library Search and also through web search engines, unless permission has been granted by the University to restrict access for a period of time. (Md Ashiqul Alam) Date: Declaration Page iii Acknowledgements Firstly, I thank and express my gratitude to the Almighty Allah for His blessings and provide me the strength to successfully finish the study. I like to express my deepest gratitude and respect to my principle supervisor Dr. Joan Kelly for her guidance, assistance, and encouragement from the very first day of my PhD till the day of submission. I am grateful for the knowledge and expertise she shared with me during my entire candidature. Her kindness and patience to my silly queries as well as advice on personal matters were remarkable. I am very fortunate to complete my PhD under her supervision and I will be ever grateful to her. I would like to acknowledge my co-supervisor Dr. Michael Lardelli for his guidance and encouragements to start this PhD program. Special thanks to Dr. Chris, Koon Ho Wong and his lab members (Dr. Yingying and Liguo) for the ChIP-seq experiments and bioinformatics analysis. I would like to thank Dr. Robin Lockington for his comments and suggestions on this thesis. I would like to thank all past and present members of Kelly lab, Dr. Jai, Dr. Vivian, and Adrian for all the discussions, advice, and encouragements. I would also like to appreciate past and present members of Lardelli lab, and Zeeshan and Rashid for their generous friendship. I would like to acknowledge the support I have received from all the members of the school including casual work at TSU with two wonderful persons, Karen and Jess, a big thank to them as well. I acknowledge the financial support that I have been provided through a Commonwealth Postgraduate Research Award and the Norman and Patricia Polglase Scholarship. I would like to express my special thanks to my parents, parents-in-laws, brothers and all relatives for their continuous support and inspirations to complete this PhD. Huge thanks to one of my mentors Dr. Miraz Rahman for his guidance and advice from my undergrad to til now. Finally, I would like to thank my wife (Sabin), daughter (Adifaah) and son (Anzar) for their love, encouragements, and support throughout the entire PhD period. Acknowledgement Page iv List of Publications The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans. Alam M.A., Kamlangdee N. & Kelly, J.M. Current Genetics (2016) DOI: 10.1007/s00294-016-0643-x Proteins interacting with CreA and CreB in the carbon catabolite repression network in Aspergillus nidulans. Alam M.A. & Kelly, J.M. Current Genetics (2016) DOI: 10.1007/s00294-016-0667-2 List of Publications Page v Chapter 1 1. Introduction and Literature Review 1.1 Introduction Eukaryote microorganisms have a complex physiology and metabolism similar to plants and animals and derive their energy from a variety of different substrates. Using this energy, they synthesize DNA, RNA, proteins, polysaccharides, vitamins and other metabolites which are essential for their survival. They show great metabolic diversity and adaptation to different environmental conditions by producing enzymes which are required for the utilization of preferred nutrient sources and by preventing the production of other enzymes which are needed to utilize less preferred nutrient sources.
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