Molecular Biology Commons^™

Protein Trafficking In The Endoplasmic Reticulum Mediated By A Tpr-Containing Adapter Protein, Nathan P. Canniff

Doctoral Dissertations

The endoplasmic reticulum (ER) is a large, multifunctional organelle that acts as the entrance into the secretory pathway, which accounts for the folding and maturation of approximately one third of the human proteome. It is the largest organelle in most cell types and is comprised of a single lumen and a contiguous membrane. The ER is responsible for a multitude of roles including protein translocation, folding, maturation, quality control, and glycosylation to name a few. These processes are buoyed by a large collection of chaperones and cochaperones, the largest subfamily of which is characterized by the presence of tetratricopeptide repeat …

A Novel Transmembrane Ligand Inhibits T Cell Receptor Activation, Yujie Ye

Doctoral Dissertations

T lymphocytes (T cells) play essential roles in the adaptive immune system. Each mature T cell expresses one type of functional T cell receptor (TCR). The TCR recognizes antigens bound to the major histocompatibility complex (MHC) in antigen presenting cells. The resulting stimulation signal crosses the transmembrane domain of TCR and initiates downstream signaling cascades. The human immune system relies on TCRs to recognize a variety of pathogens. Normally, TCR can distinguish the self-antigens from pathogenic antigens. However, dysfunction or aberrant expression of TCRs causes different inflammatory and autoimmune diseases, which afflict millions of people annually (Chapter I). Current treatments …

Characterizing The Multifaceted Roles Of The Proteasomal Deubiquitinase Uch37 In Proteostasis, Heather A. Bisbee

Doctoral Dissertations

Cellular protein pools are maintained through the biological processes of synthesis, degradation and quality control. As the dysregulation of these processes has been implicated in diseases such as neurodegeneration and cancer, understanding their functions is critical for drug development. Modification of proteins with ubiquitin may direct them to the proteasome, a large cellular protease complex, for degradation. Yet, the proteasome contains three deubiquitinating enzymes (DUBs) which remove ubiquitin from proteins, potentially altering their fate. As each DUB recognizes specific ubiquitin linkages and architectures, their activity may regulate how the proteasome handles substrates in dynamic cellular contexts. In this work, we …

Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don

Doctoral Dissertations

Circadian rhythms are 24–hour activity cycles that exist for all organisms, from yeast and bacteria to mammals. Epidemiological data has shown that disruptions to circadian rhythms are associated with various diseases, including cancers. Circadian rhythms are regulated at the cellular level by a "molecular clock,” whose oscillations in protein transcription and translation control a range of downstream pathways. However, the connections between altered rhythms and diseases at the molecular level is unclear. Therefore, in this project, I tracked circadian rhythms in a high-resolution manner and thoroughly analyzed them to more completely reflect circadian behaviors and clock functioning at the molecular …

Calmodulin Like 38 Is Required For Autophagy Of Hypoxia-Induced Cytoplasmic Rna Granules In Arabidopsis Thaliana, Sterling Field

Doctoral Dissertations

In response to the energy crisis resulting from submergence stress and hypoxia, the model plant Arabidopsis thaliana limits non-essential mRNA translation, and accumulates cytosolic stress granules. Stress granules are phase-separated mRNA-protein particles that partition transcripts for various fates: storage, degradation, or return to translation after stress alleviation. Another response by the plant cell to low oxygen stress is the induction of the turnover pathway autophagy. Stress granule regulation by autophagy occurs by a ‘granulophagy’ pathway in yeast and mammalian systems through which parts or whole stress granules are degraded. Whether this occurs in plants has not been investigated.

A connection …

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …

The Histone Variant H2av Regulates Stress Responses And Tissue Development Through Interactions With Chromatin Insulator Proteins In Drosophila Melanogaster, James Ryan Simmons

Doctoral Dissertations

The ability of a cell to sense and respond to various forms of stress is essential to maintain integrity of the genome. Numerous pathways have been implicated in cellular responses to environmental and genotoxic stresses, often involving proteins and complexes that bind DNA directly to orchestrate changes in transcription and genome organization. Chromatin insulators describe a class of protein complex that bind specific sequences in the genome and work through two classically described functions: to restrict communication between enhancers and promoters through physical separation into different genomic domains and to prevent the spread of heterochromatin into euchromatic regions of the …

The Role Of Med31 And Med12 In Directing Adipogenesis Of Human Adult-Derived Mesenchymal Stem Cells, Joseph Straub

Doctoral Dissertations

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multisubunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules: head, middle, tail, and kinase. In our introduction, we show the state of the field of Mediator study with a focus on the critical kinase module. In the following chapters, we used siRNA knockdowns to investigate the roles of the highly-conserved core subunit MED31 and the kinase module …

Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois

Doctoral Dissertations

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …

Iojap: Morphological And Physiological Phenotype Characterization In Arabidopsis, Thomas Jay Payne

Doctoral Dissertations

IOJAP protein is found in all organisms that contain a ribosome of bacterial origin. The majority of studies suggest that IOJAP plays a role in translation, although this has yet to be thoroughly investigated in plants. Using Arabidopsis thaliana, an extensive phenotype characterization of iojap mutants was performed. Many processes of plant growth were slightly impaired at optimal temperature (22˚C) but became severely hindered at low temperature (12˚C and 4˚C). These cold temperature defects manifested in an overall reduction of plant growth as well as variegation, chlorosis, leaf hyponasty, as well as reduced maximum quantum yield (F_v/F …

Involvement Of Calcium-Dependent Protein Kinases And Phosphatases In Sperm Capacitation-Associated Events, Bidur Paudel

Doctoral Dissertations

ABSTRACT To acquire fertilizing ability, mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation^1,2. At the molecular level, capacitation is associated with a fast bicarbonate (HCO₃^-)-dependent activation of a unique type of soluble adenyl cyclase (sAC) and a consequent increase in cyclic AMP (cAMP) levels and PKA activation³. Activation of a cAMP/PKA pathway results in the phosphorylation of PKA substrates, which in turn initiates activation of several signaling cascades ultimately leading to an increase in phosphorylation on tyrosine residues (P-Tyr) of sperm axonemal proteins^4,5. Increase in …

Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham

Doctoral Dissertations

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into several functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Nine TPR-containing proteins have been shown to localize to the ER and control protein organization and …

Identifying Functional Components Of The Endoplasmic Reticulum Quality Control And Degradation Factor Edem1, Lydia Lamriben

Doctoral Dissertations

The ER Degradation-Enhancing Mannosidase-Like protein 1 (EDEM1) is a critical endoplasmic reticulum (ER) quality control factor involved in identifying and directing non-native proteins to the ER-associated protein degradation (ERAD) pathway. However, its recognition and binding properties have remained enigmatic since its discovery. Here we provide evidence for an additional redox-sensitive interaction between EDEM1 and Z/NHK that requires the presence of the single Cys on the α-1 antitrypsin ERAD clients. Moreover, this Cys-dependent interaction is necessary when the proteins are isolated under stringent detergent conditions, ones in which only strong covalent interactions can be sustained. This interaction is inherent to the …

The Interplay Between Polarity Regulators, Calcium, And The Actin Cytoskeleton During Tip Growth, Carlisle Bascom Jr

Doctoral Dissertations

Plant cell growth is a meticulously regulated process whereby the cell wall is selectively loosened to allow for turgor-pressure driven expansion. The rate of expansion must equal delivery of new material, or the cell will lyse. In many plant cells, this process happens diffusely around the cell. However, a number of plant cells have anisotropic shapes that require exquisite spatial control of secretion. One simple example of anisotropic patterning is tip growth; highly polarized cell expansion utilized by pollen tubes, root hairs, and moss protonemata. Investigating the role various molecules have in tip growth sheds light on how plant cells …

Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu

Doctoral Dissertations

During development, metaphase spindles undergo large movement and/or rotation to determine the cell division axis. While it has been shown that spindle translocation is achieved by astral microtubules pulling and/or pushing the cortex, how metaphase spindle stability is maintained during translocation remains not fully understood. In budding yeast, our lab has previously proposed a model for spindle orientation wherein the mitotic spindle protein She1 promotes spindle translocation across the bud neck by polarizing cortical dynein pulling activity on the astral microtubules. Intriguingly, She1 exhibits dominant spindle localization throughout the cell cycle. However, whether She1 has any additional role on the …

Examining Shsp-Substrate Capture And Chaperone Network Coordination Through Cross-Linking, Keith Ballard

Doctoral Dissertations

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures …

Clpxp Functions In Caulobacter As A Universal And Species-Specific Protease, Robert Vass

Doctoral Dissertations

Proteolysis shapes many aspects of cellular survival, including protein quality control and cellular signaling. Powered proteolysis couples ATP hydrolysis with a degradation force that actively probes and interrogates the protein population. ClpXP, exemplifies a conserved two-part protease system charged with powered proteolysis. This protease exists as a regulatory element (ClpX), and a compartmentalized, self-contained peptidase element (ClpP). In Caulobacter crescentus, ClpXP degradation plays a crucial role maintaining proteins that exhibit proper activity, and also triggers the start of cellular differentiation. Substrate elimination requires shared aspects of the protease from both quality control and precision protein destruction functions. Here, the regulatory …

Dissecting Molecular Pathways That Ensure Proper Chromosome Segregation And Cell Division, Anna Ye

Doctoral Dissertations

Equal segregation of the genome is a prerequisite for cell survival. During cell division the duplicated DNA is compacted into chromosomes and a multi-protein macrostructure, known as the kinetochore (Kt), is assembled on each copy of compacted DNA. Simultaneously, the mitotic spindle, which is made up of microtubules (MTs), is built to facilitate the equal distribution of the chromosomes between the resulting daughter cells. Kinetochores mediate the interaction between the MTs and the chromosomes, properly positioning them for segregation. To ensure that the DNA is equally divided in every cell division, cells have built a surveillance system to detect any …

Regulated Proteolysis Of Dnaa Coordinates Cell Growth With Stress Signals In Caulobacter Crescentus, Jing Liu

Doctoral Dissertations

DNA replication is an essential process in all domains of life. Replication must be precisely regulated, especially at the step of initiation. In bacteria, the replication initiator DnaA is regulated by multiple post-translational regulations to ensure timely replication. Caulobacter crescentus has the most strict replication regulation that DNA only replicates once per cell cycle, and proteolysis of DnaA identified in this species is the only irreversible way to inhibit DnaA, suggesting it might be pivotal to restricting DNA replication. However, the responsible protease(s) and mechanism for its degradation remain unclear since its first discovery in 2005. In this thesis, I …

The Key Question In Symbiotic Nitrogen Fixation: How Does Host Maintain A Bacterial Symbiont?, Onur Oztas

Doctoral Dissertations

The fact that plants cannot use nitrogen in the gaseous form makes them dependent on the levels of usable nitrogen forms in the soil. Legumes overcome nitrogen limitation by entering a symbiotic association with rhizobia, soil bacteria that convert atmospheric nitrogen into usable ammonia. In root nodules, bacteria are internalized by host plant cells inside an intracellular compartment called the symbiosome where they morphologically differentiate into nitrogen-fixing forms by symbiosome-secreted host proteins. In this project, I explained the host proteins required to maintain bacterial symbionts and described their delivery to the symbiosome. I showed that the SYNTAXIN 132 (SYP132) gene …

Calmodulin-Like Protein 38: A Component Of Ribonucleoprotein Particles During Hypoxic Stress Responses In Arabidopsis, Ansul Lokdarshi

Doctoral Dissertations

Waterlogging stress leads to a crisis in energy metabolism and the accumulation of toxic metabolites due to the hypoxic and/or anoxic environment associated with this condition. To respond and adapt to this situation, higher plants employ an integrated genetic program that leads to the induction of anaerobic response polypeptide genes that encode metabolic and signaling proteins involved in altering metabolic flow and other adaptive responses. The study presented here shows that the Arabidopsis thaliana calmodulin-like protein CML38 is calcium sensor protein that serves as a member of the core anaerobic response gene family and is involved in modulating the survival …

The Role Of Nag-1 In Tumorigenesis, Kyung-Won Min

Doctoral Dissertations

This dissertation explores the nature of a divergent member of the Transforming Growth Factor-β [beta] superfamily, the non-steroidal anti-inflammatory drugs activated gene (NAG-1), as it relates to its regulation and biological activity in cancer context. Our lab has extensively studied on the molecular mechanism by which phytochemicals and NSAIDs induce apoptosis correlation with NAG-1 expression in human colorectal cancer (CRC) cells. Significant data from in vitro studies suggest that NAG-1 has an anti-tumorigenic activity which elicits apoptosis in a cyclooxygenase (COX)-independent manner in CRC cells. Indeed, NAG-1 transgenic mice developed less aberrant polyp foci (APC) compared to those of control …

Chromatin Insulators: Master Regulators Of The Eukaryotic Genome, Todd Andrew Schoborg

Doctoral Dissertations

Proper organization of the chromatin fiber within the three dimensional space of the eukaryotic nucleus relies on a number of DNA elements and their interacting proteins whose structural and functional consequences exert significant influence on genome behavior. Chromatin insulators are one such example, where it is thought that these elements assist in the formation of higher order chromatin loop structures by mediating long-range contacts between distant sites scattered throughout the genome. Such looping serves a dual role, helping to satisfy both the physical constraints needed to package the linear DNA polymer within the small volume of the nucleus while simultaneously …

Role Of Retinoids In The Regulation Of Hepatic Glucose And Lipid Metabolism, Rui Li

Doctoral Dissertations

The liver plays an important role in controlling glucose and lipid homeostasis. Metabolic abnormalities such as obesity and type 2 diabetes are often associated with profound changes in the expression of genes involved in hepatic glucose and lipid metabolism. Dietary nutrients provide us with macronutrients for energy and micronutrients for maintenance of general health. However, the effects of individual micronutrients on the development of metabolic diseases are unknown. Sterol regulatory element binding protein-1c (SREBP-1c) is the master regulator of fatty acid synthesis, and glucokinase (GK) is the key enzyme in glucose metabolism. Based on the preliminary results from our laboratory …

Experimental And Computational Analysis Of Chloroplast Transit Peptide Domain Architecture And Function, Prakitchai Chotewutmontri

Doctoral Dissertations

The Majority of chloroplast proteins are nuclear-encoded and utilize an N-terminal transit peptide (TP) to target into chloroplasts via the general import pathway. Bioinformatic and proteomic analyses provide thousands of predicted TPs, which show low sequence similarity. How the common chloroplast translocon components recognize these diverse TPs is not well understood. Previous results support either sequence- or physicochemical-specific recognitions. To further address this question, a reverse sequence approach was utilized such that the reverse TP contains the same amino acid composition as wild-type TP but lack similar sequence motifs. Using both native and reverse TPs of the two well-studied precursors, …

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …

Suppression Of Chronically Induced Breast Carcinogenesis And Role Of Mesenchymal Stem-Like Cells, Kusum Rathore

Doctoral Dissertations

Sporadic breast cancers are mainly attributable to long-term exposure to environmental factors, via a multi-year, multi-step, and multi-path process of tumorigenesis involving cumulative genetic and epigenetic alterations in the chronic carcinogenesis of breast cells from a non-cancerous stage to precancerous and cancerous stages. Epidemiologic and experimental studies have suggested that various dietary compounds like green tea and grape seed may be used as preventive agents for breast cancer control. In this research, I have developed a cellular model that mimics breast cell carcinogenesis chronically induced by cumulative exposures to low doses of environmental carcinogens. I used the chronic carcinogenesis model …

Novel Constitutively Active Point Mutations In The Nh2 Domain Of Cxcr2 Capture The Receptor In Different Activation States, Giljun Park

Doctoral Dissertations

Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to …

Functional Analysis Of Chromodomain Helicase Dna Binding Protein 2(Chd2) Mediated Genomic Stability, Sangeetha Rajagopalan

Doctoral Dissertations

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. …

Reverse Genetic And Cell Biological Approaches To The Study Of Developmental Functions Of Class Xi Myosin In Arabidopsis Thaliana, Eunsook Park

Doctoral Dissertations

Myosin proteins function as molecular motors that drive the ATP-dependent movement of cellular components along actin filaments. Vascular plants encode two different types of myosin, referred to as class VIII and class XI. Although class XI myosins have been suggested to function in organelle movement and cytoplasmic streaming, little is known about their cellular function in detail.

The Arabidopsis genome encodes 13 class XI myosin genes. The reasons for the relatively large number of myosin XI isoforms present within a single plant species are unknown. To investigate the function of these gene products in the cell, we determined the spatial …