Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Brain-derived neurotrophic factor (BDNF) gene : no major impact on antidepressant treatment response

The brain-derived neurotrophic factor (BDNF) has been suggested to play a pivotal role in the aetiology of affective disorders. In order to further clarify the impact of BDNF gene variation on major depression as well as antidepressant treatment response, association of three BDNF polymorphisms [rs7103411, Val66Met (rs6265) and rs7124442] with major depression and antidepressant treatment response was investigated in an overall sample of 268 German patients with major depression and 424 healthy controls. False discovery rate (FDR) was applied to control for multiple testing. Additionally, ten markers in BDNF were tested for association with citalopram outcome in the STAR*D sample. While BDNF was not associated with major depression as a categorical diagnosis, the BDNF rs7124442 TT genotype was significantly related to worse treatment outcome over 6 wk in major depression (p=0.01) particularly in anxious depression (p=0.003) in the German sample. However, BDNF rs7103411 and rs6265 similarly predicted worse treatment response over 6 wk in clinical subtypes of depression such as melancholic depression only (rs7103411: TTgenetic variation in BDNF and antidepressant treatment response or remission. Post-hoc analyses provide some preliminary support for a potential minor role of genetic variation in BDNF and antidepressant treatment outcome in the context of melancholic depression.