Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 116-125).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 99-114.

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. / Microsoft� Word 2010 / Adobe Acrobat 9.54 Paper Capture Plug-in

Repairing DNA damage is brought about by highly specific proteins that partake in a variety of DNA repair. Two of the most common types of damage are double-strand breaks (DSBs) and interstrand crosslinks. A single DSB or crosslink can potentially kill a cell if it is not repaired~ In human and other vertebrate cells, DSBs are repaired by two different mechanisms. The nonhomologous end-joining pathway can bring together the broken ends and join them, usually with the loss of some nucleotide sequence. A second pathway, homologous recombinational repair (HRR), is equally important. This repair process utilizes the information provided by another DNA molecule to restore damaged DNA. This molecule is usually a sister chromatid arising from DNA replication. This process is essentially error-free, unlike the end-joining process. Some HRR activity is required for proliferating cells to remain viable. The central protein player is RAD51, which with the help of other proteins such as XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D, performs the critical initiating steps of homologous pairing and strand transfer. The proteins encoded by the familial breast cancer genes, brcal and brca2, also play an important role in HRR. My project is concerned with studying proteins that interact with Drosophila melanogaster (XRCC2). Proteins interacting with DmXRCC2 were identified by using a yeast two hybrid system. "Bait fusion protein" (DmXRCC2 linked to GAIA BD) was constructed by Dr. Wrischnik. Tanya Dimetrijevich, a graduate student, used this bait to fish for interacting or "target" proteins. About 50 such proteins were found. I began validating these target proteins with the intention of exploring novel interactions and functions of DmXRCC2. The process of validating proteins interacting with DmXRCC2 yielded two very interesting candidate proteins-CaBPl and FAF. · CaBPl, also called protein disulfide isomerase P5, is an endoplasmic-reticulum calciumbinding protein. FAF belongs to a large family of deubiquitinating enzymes that cleave ubiquitin-protein bonds and play diverse roles in the ubiquitin pathway. One of the implications of such discoveries could be to compare and contrast DmXRCC2 and human XRCC2 in terms of their interactions and functions.

Selective protein degradation by the ubiquitin-proteasome system is essential for cellular homeostasis and the regulation of diverse biological processes. The selectivity of this system is imparted by hundreds of ubiquitin ligases that specifically recognise substrates and catalyse their ubiquitination, thereby targeting them for degradation. Among ubiquitin ligases, multisubunit cullin-RING ubiquitin ligases constitute the largest group. However, despite significant advances in understanding their assembly, regulation, and molecular architecture, the substrates and functions of most of them remain unknown. This thesis focuses on two ubiquitin ligases from the cullin-RING ubiquitin ligase 4 (CRL4) subfamily: CRL4DCAF4 and CRL4DCAF12 . To identify their candidate substrates and to address their biological roles, several different approaches have been employed. First, proteomic screening revealed a wide range of candidate substrates. Next, detailed characterisation of the identified interactions and exploration of the condition under which candidate substrates undergo degradation was performed. Finally, knockout human cell lines and mice with a targeted disruption of genes encoding DCAF4 and DCAF12 were generated to explore the physiological roles of CRL4DCAF4 and CRL4DCAF12 . In summary, the herein...

Background: Oxidative stress due to reactive oxygen species (ROS) production is a key factor in the development of heart failure (HF). This study investigated the thioredoxin (Trx) system, which plays a major role in antioxidant defense, in patients suffering from ischemic (ICM) or dilated (DCM) cardiomyopathy. Methods and results: Myocardial tissue from ICM (n = 13) and DCM (n = 13) patients, as well as septal tissue of patients with aortic stenosis but without diagnosed hypertrophic cardiomyopathy or subaortic stenosis (control; n = 12), was analyzed for Trx1, Trx-interacting protein (TXNIP) and E3 ligase ITCH (E3 ubiquitin-protein ligase Itchy homolog) expression. Trx-reductase 1 (TXNRD1) amount and activity, cytosolic cytochrome C content, and apoptosis markers were quantified by means of enzyme-linked immunosorbent assay and multiplexing. Compared with control samples, ITCH and Trx1 expression, TXNRD1 amount and activity were reduced and TXNIP expression was increased in ICM (ITCH: P = .013; Trx1: P = .028; TXNRD1 amount: P = .035; TXNRD1 activity: P = .005; TXNIP: P = .014) but not in DCM samples. A higher level of the downstream apoptosis marker caspase-9 (ICM: 582 ± 262 MFI [P = .995]; DCM: 1251 ± 548 MFI [P = .002], control: 561 ± 214 MFI) was detected in DCM tissue. A higher expression of Bcl-2 was found in DCM (P = .011). Conclusion: The Trx system was impaired in ICM but not in DCM. ITCH appeared to be responsible for the down-regulation of the Trx system. ROS-induced mitochondrial instability appeared to play a role in DCM.

The pathological changes of ubiquitination and deubiquitination following myocardial infarction (MI) and chronic heart failure (CHF) have been sparsely examined. We investigated the expression of muscle-specific E3 ubiquitin ligases and deubiquitinases in MI and CHF. Therefore, mice were assigned to coronary artery ligation for 3 days or 10 weeks as well as for sham operation (each n = 10). Expression of E3 ligases (MAFBX, MURF1, CHIP, ITCH, MDM2) and deubiquitinases (A20, CYLD, UCH-L1, USP14, USP19) was determined. After MI and in CHF, the mRNA expression of MURF1, CHIP and MDM2 (all p < 0.05) was decreased. Protein expression analyses revealed that ITCH expression decreased in CHF (p = 0.01), whereas MDM2 expression increased in MI (p = 0.02) and decreased in CHF (p = 0.02). Except for USP19 mRNA expression that decreased at 3 days and 10 weeks (both p < 0.01), the expression of other deubiquitinases remained unaffected after MI and CHF. The expression of myocardial E3 ligases is differentially regulated following MI, raising the question of whether an upstream regulation exists that is activated by MI for tissue protection or whether the downregulation of E3 ligases enables myocardial hypertrophy following MI.