No description available.

No description available.

No description available.

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl isomerase enzyme (PPIs) that catalyze the cis-trans inter-conversion of peptidyl-prolyl bonds in proteins. This non-covalent post-translational modification is a reversible mechanism to modulate protein structure and function. PPIs have been implicated in a wide variety of processes from protein folding to signal transduction. Despite these enzymes being ubiquitous, the substrates and functions of most PPIs have yet to be described. FKBP25 is a nuclear FKBP that has been shown to associate with transcription factors and chromatin modifying enzymes, however its functions and substrates remain largely unresolved. FKBP25 is the human ortholog of S. cerevisiae Fpr4, which has been shown to regulate the chromatin landscape by two distinct mechanisms: 1. Acting as a histone chaperone at ribosomal DNA, and 2. Isomerizing histone prolines. Based on these observations, I hypothesized FKBP25 regulates chromatin and/or ribosome biogenesis through isomerization of histone prolines and a discrete collection of substrate proteins. While small molecule inhibitors exist for FKBPs, applying them to dissect the specific function(s) of any given FKBP is confounded by the fact that multiple FKBPs are found in each organism, and several are inhibited by these molecules. In Chapter 2, I biochemically and structurally characterize a set of FKBP25 loss-of-function mutants, yielding a toolset capable of distinguishing between catalytic and non-catalytic functions. These reagents provide the tools necessary to analyze potential substrates of FKBP25 identified in my research going forward. In Chapter 3, I present the first unbiased proteomic screen of FKBP25 associated proteins and show that it interacts with a large number of ribosomal proteins, ribosomal processing factors and a smaller subset of chromatin proteins. I focus on the interaction between FKBP25 and nucleolin, a multi-functional nucleolar protein, and show that FKBP25 interacts with nucleolin and the pre-60s ribosomal subunit in an RNA dependent fashion. In Chapter 4, I gain insight into the role of FKBP25 in ribosome biology, and demonstratex that FKBP25 regulates RNA binding activity of nucleolin, however this does not appear to involve cis-trans prolyl isomerization. Collectively, my work establishes FKBP25 as the first human FKBP to be implicated in the maturation of the pre-60S ribosomal subunit in the nucleus. My data supports a model whereby FKBP25 associates with the assembling large ribosomal subunit, where it is likely to chaperone protein-RNA interactions. / Graduate

Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171

No description available.

Abstract not available

Includes bibliographical references (leaves 291-311)

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. The result of RAGE-ligand interactions augments the proinflammatory mechanisms acting in chronic inflammatory diseases. RAGE recognises a wide range of ligands that have no apparent structural similarities. It is unclear what controls this promiscuity of RAGE. The extracellular domain of RAGE has two potential glycosylation sites. It is speculated that N-linked glycosylation may have significant impact on ligand recognition, especially of S100 calcium binding protein ligands. Two objectives of this thesis were to establish whether S100A9 acts as a ligand for RAGE and to investigate whether glycosylation of RAGE has any influence on ligand recognition. These were achieved by generating two forms of RAGE. HEK 293 cells were transfected to express full-length, membrane-bound RAGE or a secreted form comprising the extracellular domain of RAGE. Site-directed mutagenesis of RAGE showed that asparagine at position 25 is the pre-dominant N-linked glycosylation site. The carbohydrate added to asparagine 25 was further modified to a non-sialylated carboxylated N-linked glycan, specifically recognised by monoclonal antibody GB 3.1. Binding studies showed that different RAGE ligands have individual requirements for glycosylation of the receptor. Binding of AGE-modified AGE-BSA or of S100B to RAGE occured independent of N-linked glycosylation of the receptor. RAGE also binds the S100 protein, MRP-14 (S100A9). In contrast to AGE-BSA or S100B, the non-sialylated carboxylated N-glycan expressed on RAGE is crucial for binding to MRP-14. However, RAGE produced in tunicamycin containing medium and thus lacking N-linked glycosylation, shows strong binding to MRP-14. It was concluded that two forms of binding are involved: the first mechanism relies on the non-sialylated carboxylated N-glycan attached to RAGE and acts in a "tethering" fashion. The second mechanism involves a conformational change of RAGE, which results in exposure of a binding site(s) and a more conventional receptor-ligand interaction. Another objective for this thesis is to study the expression of RAGE and its alternatively spliced variants. PCR analysis has revealed several variants of RAGE that result from alternative splicing mechanisms. The variant proteins are soluble due to a lack of membrane localising sequence. PCR results confirmed the presence of transcripts encoding for spliced variants of RAGE in several tumour cell lines. Among these were transcripts that should encode a soluble form of sRAGE 2. Furthermore, it was shown that sRAGE 2 transcript can be present in forms that contain the ligand-binding V-domain of RAGE or that are N-truncated and lack the V-domain. This is the first report of a soluble, N-truncated sRAGE 2 variant. The results in this thesis add to our knowledge of RAGE biology. MRP-14 (S100A9) is identified as a new ligand. The control of MRP-14/RAGE interaction relies on N-linked glycosylation of the receptor and further modification of the carbohydrate. "Tethering" or stronger receptor-ligand interactions are suggested as mechanisms for controlling RAGE recognition of multiple ligands. Soluble RAGE variants that lack or contain V-domain binding regions, and hence sites for glycosylation were produced. These have the capacity to compete with membrane-bound receptor for available ligand. The control of the expression of soluble RAGE variants, in concert with the control of various modification to carbohydrate expressed on the receptor, adds a level of complexity to ligand specificity. This may ultimately result in different paradigms of the inflammatory process.

Includes bibliographical references (leaves 291-311) / xxiii, 313 leaves : ill. (chiefy col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002