<a>The 20S proteasome, a multi-subunit protease complex, present in all domains of life and some orders of bacteria, is involved in degradation of the majority of cellular proteins. Structurally, it is made of α and β subunits arranged in four heptameric rings, with inner two β-rings sandwiched between outer two α-rings. The 20S proteasome in prokaryotes usually has one type of α and one type of β subunits, whereas eukaryotes have seven distinct types of α and seven distinct types of β subunits. Unlike the highly conserved structure of proteasome, its assembly pathway is different across the domains. In archaea and eukaryotes, proteasome assembly begins with α subunit interactions leading to the α-ring formation. By contrast, bacterial proteasome assembly pathway bypasses the α-ring formation step by initiating assembly through an α and β subunit interaction first. These early interactions are not well understood due to their highly rapid and dynamic nature. This dissertation focused on understanding the early events in proteasome assembly and contributed three significant findings. First, the archaeal proteasome assembly can also begin without formation of α-rings, demonstrating the coexistence of a bacterial-like assembly pathway. Second, a novel assembly intermediate was identified in yeast, and its composition argues for the presence of a similar α-ring independent assembly pathway. Third, the assembly chaperone Pba3-Pba4 prevents the formation of high molecular weight complexes arising from spontaneous and non-productive interactions among the α subunits. These findings provide a broader understanding of proteasome biogenesis and suggest considering proteasome assembly event as a network of interactions rather than a linear pathway. The results also shed light on assembly chaperone’s contribution in increasing the efficiency of proteasome assembly by streamlining the productive interactions.</a>

Arachidonylethanolamide (AEA) is a bioactive lipid ligand for mammalian cannabinoid receptors (CB). Thus far, AEA was reported to occur only in animals and was shown to regulate a wide range of physiological responses. Our recent fi nding of the occurrence of AEA in moss has led us hypothesize that AEA might mediate stress responses in plants, similar to that in animals. In mammals, AEA is generated from hydrolysis of N-acylphosphatidylethanolamine (NAPE) by a NAPE-specifi c phospholipase D (NAPE-PLD), and degraded by a fatty acid amide hydrolase (FAAH) and this metabolic pathway is highly conserved among eukaryotes. Here, using in silico approach, putative genes encoding for AEA pathway enzymes, were identifi ed in moss. Full-length coding sequences for putative NAPE-PLD and FAAH were isolated from Physcomitrella patens and were cloned and expressed into a heterologous expression vector. Biochemical characterization of AEA pathway enzymes is underway and is expected to lead to generation of AEA metabolite mutants in moss. Such mutants will allow for elucidation of the role of AEA in development of moss and mediating stress responses. Overall, this study will provide novel insights into functional and evolutionary role of lipid-mediated signaling in plants.

N-Acylethanolamines (NAEs) including anandamide (NAE 20:4) are fatty acid ethanolamides generated by the hydrolysis of N-acylphoshotidylethanolamine (NAPE) by phospholipase D (PLD) and degraded by fatty acid amide hydrolase (FAAH). In mammals, ligands such as NAE 20:4 act through cannabinoid receptors and regulate several physiological processes like neuroprotection, pain perception, mental depression, and appetite suppression. In plants, NAE with chain length C12 to C18 are common and affect physiological processes such as cytoskeletal organization, endomembrane trafficking, cell wall and cell shape formation, seedling growth and response to stress. However, our recent identification of NAE 20:4 in moss, Physcomitrella patens prompted us to elucidate its metabolic pathway and physiological implications. We hypothesize that unique NAE metabolites such as anandamide in moss might play a role in rendering moss its ability to tolerate temperature, dehydration, salt and osmotic stress. To address the above hypothesis, three main objectives are being pursued using P patens. 1)Biochemical and molecular characterization of NAE metabolic pathway, 2) Generation and phenotypic characterization of NAE metabolite mutants, and 3) Elucidation of the physiological role of NAEs in abscisic acid-mediated dehydration tolerance. A NAPE-PLD, known to synthesize NAE 20:4 has been identified in mammals and FAAH in several eukaryotes, including plants. Here, identification and cloning of putative NAPE-PLD and FAAH genes that are likely involved in NAE synthesis and degradation, respectively, in P patens is discussed. Our long-term objective is to understand lipid-mediated stress responses in plants.

No description available.

A biological pathway is a series of reactions and molecular interactions. Pathway information provides a blueprint for biomedical researchers to devise new treatment and diagnostic solutions for human diseases. Pathway data are publicly available in many databases. Most of the databases however have only partial coverage of human biological pathways, especially for signal transduction and gene regulatory pathways. Comprehensive knowledge and a combined view for all types for biological pathways may lead to insights into the molecular physiology of cells and drug discovery. In this project, we collected human signaling pathway data from different database sources. The pathway data comes from the Biocarta, Protein Lounge, Resnet and NCI-Nature Curated databases. We analyzed the structures of collected data, and developed a comprehensive data model to manage integrated information of molecules, complexes, regulation relationships of molecules, and reactions involved in signaling and regulatory pathways. The integrated database, the Human Pathway database (HPD), is a data warehouse of a comprehensive collection of biological reactions, regulators, and metabolites, serving as a potential platform for future pathway analysis studies. We developed the database to manage integrated biomolecular information related to pathways. In HPD, which uses Oracle 10g, there are a total of 1,895 pathways, 10,631 molecular entities (proteins, complexes and compounds) and 4,370 reactions that were consolidated and integrated into a single relational database platform using Oracle 10g. We also developed a prototypical GUI for the navigation and querying of biological pathways. Such a system, when completed, and used in conjunction with future pathway data visualization and analysis tools, could provide a framework for systems biology studies. We developed HPD with comprehensive annotation data. For example, the kinase - disease association information and perturbation effect of several environmental factors are unique in HPD. The potential for merging similar pathways—“pathway mergability” —was created based on gene/protein identifiers and to provide with non-redundant pathway information. To test the technology we have done two case studies. The first case study addressed the Alzheimer’s disease by coupling pathway network analysis with gene expression data. The second case study addressed the cellular responses by coupling the pathway network analysis with protein expression data. These case studies demonstrate how an integrated pathway database can be used to generate new insights into the discovery of biomarkers, and extend our understanding of cellular physiology.

The number and complexity of genome-scale metabolic networks is increasing as new systems are characterized and existing models are extended. Tools for visualization of network topology and dynamics are not keeping pace and are becoming a bottleneck for advancement. Specically, visualization tools are not optimized for human comprehension and often produce layouts where important interactions and inherent organization are not apparent. Researchers seek visualizations in which the network is partitioned into functional modules and compartments, arranged in linear, cyclic, or branching schema as appropriate, and most importantly, can be customized to their needs and shared. Challenges include the wide diversity in the biological standards, layout schemas, and network formats. This work introduces a web-based tool that provides this functionality as an extension to the existing web-based tool called Pathway Pioneer (www.pathwaypioneer.org). Pathway Pioneer is a dynamic web-based system built as a front-end graphical user interface to the ux balance analysis tool COBRA-py. Full click-and-drag layout editing capabilities are added allowing each metabolite and reaction to be translated and rotated as connecting edges are automatically redrawn. Initial automated layouts for new models maximize planarity while clustering reactions based on subsystem module and compartment. The users are given maximum exibility to design specific layouts while details of convention, such as joined in and out of reaction edges, disconnected co-factors, and connected metabolites, are automatically handled. Layouts can be shared among researchers and explored to archival Symphony format, along with pdf and png images. This tool provides the user with a semi automatic layout algorithm along with graphical and interactive tools to fully customize the network layout for optimal comprehension. Export capabilities are compatible with COBRA-py and other visualization tools. It provides a platform for share model development and innovation to the community, sharpening the R&D curve, and improving the turn-around time of model reconstruction at the genome-scale. Pathway Pioneer provides unique capabilities in customization of metabolic networks that complements and overcomes limitations of the growing body of existing tools.

No description available.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Type II diabetes mellitus (TIIDM) causes multiple complications under chronic hyperglycemia. Long term persistent exposure to elevated glucose conditions is considered one of the major factors for diabetic complications. Pathologically, mechanical and biochemical stimuli will induce a signaling cascade in cardiac fibroblasts, which causes myocardial fibrosis and leading to ventricular stiffness. Non-enzymatically, high levels of glucose can react with long-lived proteins, such as collagen to form advanced glycation end-products (AGEs). AGEs have been shown to be associated with many of the diabetic cardiovascular complications due to their interaction with the receptor for AGE (RAGE). AGE/RAGE activation stimulates the secretion of growth factors, promotes increased collagen production that leads to tissue fibrosis, and increased RAGE expression. The purpose of this study is to identify the role for Rap1a in regulating fibrosis under TIIDM conditions, as well as to offer insight into the AGE-RAGE signaling cascade definition for cardiovascular extracellular matrix remodeling under TIIDM condition. To test our hypothesis, both loss-ofunction and gain-ofunction based experiments were performed to manipulate Rap1a protein expression in AGE-RAGE mediated fibrosis. Also, we down-regulated the activity of downstream molecules in the AGE-RAGE signaling cascade, such as protein kinase C-ζ (PKC-ζ) and ERK1/2 by specific inhibitor treatments, to test their positions in AGE-RAGE mediated fibrosis pathway. To perform our experiment in vivo, we used high fat diet to feed Rap1a heterozygous mice in order to build a Rap1a heterozygous diabetic animal model. Our results showed that Rap1a protein plays a key role in AGE-RAGE signaling pathway under TIIDM, and changes in Rap1a activity altered the signaling pathway. Also, we found that PKC-ζ is the upstream player relatively to ERK1/2, and Rap1a is the upstream player for both PKC-ζ and ERK1/2. By understanding the role Rap1a played in AGE-RAGE signaling cascade, a new molecular mechanism is found possibly to reduce the cardiac fibrosis in TIIDM patients.

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as “capacitation” in order to be able to fertilize an oocyte. Among these, biochemical changes such as an increase in tyrosine phosphorylation of some sperm proteins have been correlated with the sperm capacity to fertilize an egg and found to be regulated by a cAMP dependent pathway. The influx of ions such as Ca2+ and HCO3- induce the activation of a soluble adenylyl cyclase (SACY) increasing the cAMP levels within the cell that leads to the activation of a protein kinase A (PKA), and a subsequent increase in protein tyrosine phosphorylation. This modification in sperm proteins seems to be essential for induction of a change in the motility pattern known as hyperactivation that enables the sperm to penetrate the zona pellucida of the oocyte and initiate fertilization. Since PKA is a serine/threonine kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Based on the finding that in somatic cells PKA activates c-Src, it has been proposed that the Src family of protein kinases (SFK) are the intermediate players involved in tyrosine phosphorylation induced by PKA activity. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activity during stallion sperm capacitation (2) To evaluate the involvement of the Src family of protein kinases (SFK) on stallion sperm phosphorylation events associated with capacitation. Standard In Vitro Fertilization (IVF) has not been reproducibly successful in the horse. Recent data indicate that good fertilization rates may be achieved after treatment of sperm with procaine to induce hyperactivation. Our objectives were also to determine if drugs used in other species as well as procaine induce hyperactivation in stallion sperm and to evaluate biochemical changes such as protein tyrosine phophorylation.