Department: Biochemistry ![[clear]](close-x.png "Remove this limiter")
81 matches in the database.
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1.
Abbas, Atheir Ibrahim.
PSD-95 Regulates Serotonin Receptor Function in vivo.
Degree: PhD, Biochemistry, 2009, Case Western Reserve University
► The 5-hydroxytryptamine 2A (5-HT2A) receptor, a target for hallucinogens and some antipsychotics,…
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▼ The 5-hydroxytryptamine 2A (5-HT2A) receptor, a target for hallucinogens and some antipsychotics, is thought to play a prominent role in regulating mood, perception, and cognition. The closely related 5-hydroxytryptamine 2C (5-HT2C)receptor is also thought to be involved in a number of central nervous system processes including mood and temperature regulation. Due to the behavioral effects that result from activation and blockade of 5-HT2A and 5-HT2C receptors, it has been suggested that these receptors can modulate glutamatergic neurotransmission, though the biochemical links between the metabotropic serotonin and ionotropic glutamate systems have remained a mystery. In the studies presented herein we show that the postsynaptic PDZ domain-containing scaffolding protein postsynaptic density protein of 95kDa (PSD-95), a 5-HT2A/2C-interacting protein, is an important biochemical link between the serotonin and glutamate systems. We show that, in the absence of PSD-95 in vivo, 5-HT2A and 5-HT2C receptor expression is reduced due to an increase in the rate of receptor turnover. We also provide evidence that targeting to the appropriate apical dendritic compartment is impaired in neurons cultured from PSD-95 knockout mice, and that lentiviral addback of PSD-95 to knockout neurons rescues targeting. We also examine signaling at both the biochemical and behavioral level. With respect to the 5-HT2C receptor, we show that the ability of a 5-HT2C agonist to induce c-fos, a marker of neuronal activation, is greatly reduced in the absence of PSD-95. We also present data showing that 5-HT2A-mediated hallucinogen-induced head twitch is also reduced in the absence of PSD-95. Finally, we provide evidence that clozapine, which is thought to correct the abnormalities in glutamatergic neurontransmission seen in some animal models of psychosis via a 5-HT2A-dependent mechanism, is unable to exert its therapeutic efficacy in PSD-95 knockout mice. Together the data presented herein provide the first biochemical link between the metabotropic serotonin and ionotropic glutamate systems. Our studies also suggest that this link is relevant not only with respect to the regulation of 5-HT2A and 5-HT2C receptor function, but also with respect to hallucinogen action and the neurochemistry underlying psychosis.
Advisors/Committee Members: Roth, Bryan.
Subjects: Biochemistry; Pharmacology
Keywords: 5-HT2A; 5-HT2C; PSD-95; hallucinogen; antipsychotic; clozapine; serotonin receptors
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2.
Aiyar, Ashok Anantharaman.
Role of RNA structures in the initiation of Rous sarcoma virus reverse transcription.
Degree: PhD, Biochemistry, 1994, Case Western Reserve University
► Roles of RNA structures in the 5′ non translated region of Rous…
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▼ Roles of RNA structures in the 5′ non translated region of Rous Sarcoma Virus (RSV) in the initiation of reverse transcription were evaluated. Besides the previously demonstrated U5-leader stem, reverse transcription of RSV was found to require the U5-IR stem. Disruption of the latter by substitution mutations could be compensated by secondary mutations that restored the structure of the stem but not its sequence. Additionally a role for U5 RNA sequences in base-pairing to the TψC loop sequences in primer tRNA Trp was established. This was examined in vivo and in vitro using a substitution mutation that abrogates the U5-TψC interaction. In vitro this mutation could be rescued by using a primer that contained sequences complementary to the substitution. Further studies revealed that these three regions of duplex RNA structure must be maintained at a specific spatial orientation with respect to each other. Disruption of "spacer" sequences between the duplex regions by insertion and deletion mutations caused defects in initiation. In contrast, substitution mutations in these non base-paired spacer sequences had no effect.
Advisors/Committee Members: Leis, Jonathan P.
Keywords: RNA structures; Rous sarcoma; virus reverse transcription
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3.
Anthony, Donald D.
Translation initiation: Typical and atypical mRNAs.
Degree: PhD, Biochemistry, 1991, Case Western Reserve University
► This thesis documents studies of ribosome-mRNA interactions during eukaryotic translation initiation. The…
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▼ This thesis documents studies of ribosome-mRNA interactions during eukaryotic translation initiation. The interaction was studied for: typical cap-dependent, atypical cap-independent (internal initiation), and atypical re-initiation mechanisms. For cap-dependent initiation, the focus was the initiation factor requirements of ribosome-mRNA binding versus ribosome scanning. The technique of primer extension inhibition was adapted from the prokaryotic system to study the eukaryotic ribosome-mRNA interaction. Ribosome-mRNA complexes for both 80S ribosomes and 40S ribosomal subunits located at the initiation codon were individually analyzed. Complexes were also isolated where the 40S subunit was located 5′ to the initiation codon, confirming the two step model of ribosome binding followed by scanning. In a reconstituted system composed of highly purified components, it was found that particular components were necessary for localizing the 40S subunit at the initiation codon (scanning), while other components were required for ribosome-mRNA binding. Specifically, ATP, GTP, Met-tRNA i, and eIF-4B were not required for the ribosome binding event, but were required for scanning to the initiation codon. The factors eIF-3, eIF-2, and eIF-4F were required to form a ribosome-mRNA complex which could be isolated by sucrose gradient centr ifugation, but was not stable enough to be analyzed by primer extension inhibition. Atypical ribosome binding events observed during cap-independent and internal initiation were examined by programming an in vitro translation system with a number of constructed bicistronic mRNAs. Results indicated that both processes occur by the same mechanism. Furthermore, initiation factor addition studies indicate that the cap-interacting initiation factor (eIF-4F) as well as eIF-4B are involved in internal binding of ribosomes on an mRNA, which in turn leads to translation of an open reading frame.
Advisors/Committee Members: Merrick, William C.
Subjects: Chemistry, Biochemistry
Keywords: Typical atypical mRNAs
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4.
Arida, Ahmad Raslan.
Lipoprotein LprG Enhances TLR2 Agonism of Mycobacterium tuberculosis.
Degree: MS, Biochemistry, 2011, Case Western Reserve University
► Toll-like receptor 2 (TLR2) agonists in the mycobacterial cell wall are important…
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▼ Toll-like receptor 2 (TLR2) agonists in the mycobacterial cell wall are important determinants of immune responses of Mycobacterium tuberculosis (Mtb). The Mtb lipoprotein LprG was found to associate with glycolipids, contributing to its TLR2 agonist activity. Isolation of LprG from mycobacteria suggests that LprG is able to bind multiple cell wall glycolipids, including lipomannan (LM), and lipoarabinomannan (LAM). We have some evidence that LprG prefers binding to LAM over LM. Experiments are ongoing to investigate the mechanism and kinetics of this preferential binding. The glycolipid chaperone role of LprG in the context of the whole bacterium TLR2 agonism was examined when individual lipoproteins were overexpressed. Overexpression of acylated LprG increased the TLR2 agonist activity of the whole bacterium; however, a non-acylated form of LprG does not increase TLR2 activity of the bacteria. We have found that LprG and its glycolipid cargo are major contributors to whole Mtb TLR2 agonist activity.
Advisors/Committee Members: Harding, Clifford.
Subjects: Biology; Immunology
Keywords: Mycobacterium tuberculosis, lipoprotein, LprG, TLR2
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5.
Bifano, Abby Lynn Shumaker.
The Role of a Nuclear-Encoded DEAD-box Protein from Saccharomyces cerevisiae in Mitochondrial Group I Intron Splicing.
Degree: PhD, Biochemistry, 2010, Case Western Reserve University
► DEAD-box proteins are a large class of ATPases that are involved in…
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▼ DEAD-box proteins are a large class of ATPases that are involved in almost all aspects of RNA metabolism. Many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acids, and to unwind and anneal nucleic acids strands and, therefore, DEAD-box proteins are generally thought to modulate RNA structure in vivo. To a large degree, however, the functions of individual DEAD-box proteins in vivo remain unclear and, furthermore, it is unknown which of the aforementioned properties are important for their activities in vivo. Thus, it is critical to define the mechanisms by which DEAD-box proteins function with their native substrates, in order to understand the permissible roles of these important enzymes in RNA metabolism and, more broadly, gene expression. The work presented here is an investigation into the role of a representative DEAD-box protein, Mss116p, and the mechanisms by which it functions in the splicing of a native group I intron substrate, the aI5β intron from the mitochondrial COX1 gene in S. cerevisiae. In the first study, we show that Mss116p, in an ATP-dependent manner, specifically facilitates the second step of aI5β splicing in vitro and that its role in exon ligation is temporally controlled and dependent on the “upstream” activity of another RNA binding protein, Mrs1p. We also demonstrate that the ability of Mss116p to efficiently hydrolyze ATP, rather than unwind a stable model duplex, is critical for its function in aI5β splicing. In the second study, we explored the catalytic properties of Mss116p that were required for the splicing of the aI5β intron, as well as additional native intron substrates, in vivo. Similar to our biochemical results, we found that efficient ATPase activity, rather than robust unwinding activity, was required for Mss116p’s role in the splicing of all examined intron substrate in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, our observations support a model in which enzyme turnover is a crucial factor in the splicing function of Mss116p.
Advisors/Committee Members: Snider, Martin.
Subjects: Biochemistry; Molecular biology
Keywords: DEAD-box proteins; group I intron splicing; catalytic RNAs; mitochondrial RNA processing; Saccharomyces cerevisiae
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6.
Black, Steven Gregory.
C-REACTIVE PROTEIN: A STUDY OF ITS FUNCTIONAL DOMAINS USING TRANSGENIC MICE.
Degree: PhD, Biochemistry, 2005, Case Western Reserve University
► C-reactive protein (CRP) is an acute phase protein in humans whose plasma…
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▼ C-reactive protein (CRP) is an acute phase protein in humans whose plasma concentration can increase 1,000x following an inflammatory stimulus. Two of the most widely studied properties of CRP are its ability to bind phosphocholine (PCh) and to activate complement. These in vitro properties of CRP have been hypothesized to play a critical role in CRP function. Initially, we generated transgenic mice expressing rabbit CRP (rbCRP) to develop an in vivo assay for rbCRP function. These animals have been used to demonstrate that transgenic rbCRP had a net anti-inflammatory effect in mouse models of inflammation which include models of endotoxemia, pulmonary alveolitis, and antigen induced arthritis. In order to understand the mechanism through which rbCRP imparts its effects, we created additional lines of transgenic mice expressing variants of rbCRP. Based on the co-crystal structure of C-reactive protein and phosphocholine, a variant of rbCRP (F66Y/E81K) was created that was incapable of binding PCh. A second variant of rbCRP (Y175A) with an altered ability to activate complement was generated based on site-directed mutagenesis studies. To test the importance of these two functional domains of rabbit CRP in vivo we utilized these mice in a model in which they were challenged with a lethal dose of a mediator of inflammation, platelet activating factor (PAF). Our results indicated mice expressing wild type and Y175A rbCRP were protected against challenge by PAF, while mice expressing F66Y/E81K rbCRP were not. PAF contains a PCH moiety, and in an in vitro solid phase binding assay, wild type and Y175A rbCRP could bind PAF, but F66Y/E81K rbCRP could not. We conclude that PCh binding is critical for the rbCRP protective effect and hypothesize that the mechanism of rbCRP protection from PAF lethality involves sequestration of PAF by rbCRP so that PAF is unable to bind its receptor. We further conclude that its ability to activate complement is not necessary for protection from PAF challenges. These studies are the first to dissect the functional domains of rbCRP in an in vivo pathophysiological setting.
Advisors/Committee Members: Samols, David.
Subjects: Biology, General
Keywords: acute phase protein; inflammation; complement; phosphocholine; transgenic mice
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7.
Bowers, Heath Andrew.
RNP Remodeling and Cofactor Modulation by the DEAD-box Protein Ded1p.
Degree: PhD, Biochemistry, 2009, Case Western Reserve University
► DEAD-box proteins are involved in virtually all aspects of eukaryotic RNA metabolism.…
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▼ DEAD-box proteins are involved in virtually all aspects of eukaryotic RNA metabolism. As members of the helicase superfamily 2 (SF2), DEAD-box proteins utilize ATP hydrolysis to unwind RNA, assemble large protein complexes on RNA, and remodel RNA protein complexes (RNPs). In the cell however, DEAD-box proteins function in the context of RNPs during processes such as splicing and translation. How the biochemical activities of DEAD-box proteins are utilized in a physiological setting is an important and central question in RNA metabolism. In this thesis, we address this issue by examining RNP remodeling and cofactor modulation by the DEAD-box protein Ded1p.We demonstrated that Ded1p did not actively displace the RNA binding proteins U1A and TRAP from their cognate RNA binding sites. Additionally, we established that the context of a RNP determined active displacement by Ded1p and propose a model for RNP remodeling by DEAD-box proteins. We found that that an inability to actively displace other proteins from RNA can provide non-sequence specific DEAD-box proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. We further identified a physiologically relevant interaction between Ded1p and the translation initiation factor eIF4G. We showed that eIF4G did not modulate Ded1p ATPase activity, but did inhibit strand separation by Ded1p. Interestingly, Ded1p greatly increased eIF4G’s affinity for RNA even to RNAs too small for eIF4G to bind alone. Our results suggest that Ded1p’s biochemical activities facilitate eIF4G RNA binding. We propose a basic model for Ded1p enhancement of eIF4G RNA binding which may be relevant for Ded1p’s role during translation initiation.
Advisors/Committee Members: Jankowsky, Eckhard.
Subjects: Biochemistry; Molecular biology
Keywords: DEAD-box proteins; DExH/D proteins; Ded1p; eIF4G; cofactor; RNPase; helicase
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8.
Cheng, Xiwen.
The Functional Study of Transcriptional Corepressor G-Protein Suppressor 2 (GPS2) and Tumor Suppressor Promyelocytic Leukemia (PML).
Degree: PhD, Biochemistry, 2010, Case Western Reserve University
► We have identified G-protein pathway suppressor 2 (GPS2) as an integral component…
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▼ We have identified G-protein pathway suppressor 2 (GPS2) as an integral component of the silencing mediator of retinoid acid and thyroid hormone receptor (SMRT) corepressor complex. Functional studies revealed that GPS2 is functionally important for estrogen receptor alpha (ERα)-mediated transcriptional regulation, and that GPS2/SMRT complex is important for maintaining normal proliferation of MCF-7 breast cancer cells. The promyelocytic leukemia protein (PML) is a well-known tumor suppressor, but its role in endothelial cells (ECs) is largely unknown, despite its high expression in endothelium and inflamed tissues. We have demonstrated that PML negatively regulates angiogenesis and cell migration, reconciling with upstream signal transducer and activator of transcription 1 (STAT1) and downstream integrin β1 (ITGB1), thus identifying a novel pathway in which PML mediates the TNFα/IFNα signaling in ECs. Finally, we carried out microarray analysis in ECs and identified the differentially expressed genes when PML is knocked down and/or when cells are treated with TNFα. We further characterized the functional ontology of these gene lists and identified groups of interesting genes that warrant future study.
Advisors/Committee Members: Kao, Hung-Ying.
Subjects: Biochemistry; Bioinformatics; Cellular biology; Molecular biology
Keywords: GPS2; SMRT; Estrogen Receptor alpha; ERα; pS2; MCF-7; ChIP; proliferation; tamoxifen resistance; PML; angiogenesis; migration; HUVEC; HMVEC; cytokine; TNFα; IFNα; ITGB1
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9.
Chen, Xuguang.
Cellular Uptake of DNA Nanoparticles and Regulation of Cell Surface Nucleolin.
Degree: PhD, Biochemistry, 2009, Case Western Reserve University
► DNA nanoparticles are in vivo gene transfer vectors in development for treating…
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▼ DNA nanoparticles are in vivo gene transfer vectors in development for treating cystic fibrosis. We previously discovered that they deliver transgenes efficiently to the mouse airway without inducing significant inflammatory cells and cytokines. Using rhodamine-labeled DNA nanoparticle, we found that they accumulate in the nucleoli in well-differentiated airway epithelial cells, colocalizing with nucleolin. We employed surface plasmon resonance (SPR) technology to demonstrate direct binding of nucleolin to DNA nanoparticles with KD = 25.9 nM. Nucleolin is expressed on the surface of HeLa cells and human tracheal 16HBEo- cells. Cell surface nucleolin shares a similar intracellular trafficking pattern with rhodamine-nanoparticles. Manipulations of nucleolin indicate that it is essential for the transfection of the nanoparticles. Moreover, purified nucleolin significantly blocks transfection, supporting the hypothesis that nucleolin is a critical receptor for the nanoparticles. We then studied the regulation of surface expression of nucleolin and found that cyclin dependent kinase 1 (Cdk1), rather than casein kinase 2 (CK2) phosphorylation, promotes its surface appearance. The N-terminus of nucleolin including the 8 consecutive Cdk sites is required for efficient surface expression. Inhibition of Cdk1 blocks the increase of surface nucleolin at G2/M phase transition. Mutations of the Cdk sites to glutamate increase its surface expression. We also found that nucleolin exists in lipid rafts on the membrane, and DNA nanoparticles can be recovered from lipid raft fractions following cellular uptake. Moreover, transfection of the nanoparticles is blocked by cholesterol depletion using drugs such as filipin and methyl-β-cyclodextrin. Nucleolin directly associates with flotillin-1, an integral lipid raft protein regulating raft-mediated endocytosis; and the association is not disrupted by cholesterol depletion. Taken together, DNA nanoparticles enter cells via lipid rafts and cell surface nucleolin, which is positively regulated by cyclin dependent kinase Cdk1.
Advisors/Committee Members: Davis, Pamela.
Subjects: Biomedical research; Cellular biology
Keywords: Cystic fibrosis; Gene therapy; non-viral vector; endocytosis; lipid raft; protein trafficking; phosphorylation; cell cycle
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10.
Chen, Yuanyuan.
Characterizing RNA Structure and synthesis by Raman Microscopy.
Degree: PhD, Biochemistry, 2010, Case Western Reserve University
► Raman microscopy has been broadly applied to the structural studies of macromolecules,…
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▼ Raman microscopy has been broadly applied to the structural studies of macromolecules, such as proteins and RNAs. Using isotopically labeled samples, we are able to identify the signals from specific regions of the molecule or the complex, so that the structural or chemical information from local regions of a big molecule or complex can be investigated. In this thesis, isotopically edited samples have been used in two different applications: 1) probing the adenine rings and linked backbone conformation of domain V (D5) of Group II intron in solution, using isotopically edited Raman spectroscopy; 2) monitoring transcription initiation in a single crystal containing N4 mini-vRNA polymerase in real-time, using time-dependent Raman microscopy. In the first case, the D5 RNA was specifically labeled with 13C and 15N on the A base rings and the linked ribose. All the A ring modes and A-linked backbone conformations are identified; helical and non-helical backbone conformations, as well as local base ring conformations are compared in the presence and absence of Mg2+; the Mg2+•••PO2- (backbone) interaction is shown to be affected differently by monovalent ions, which can be anti-correlated with the in vitro activities of the reconstructed ribozyme in the corresponding monovalent ion conditions. In the second project, the first phosphoryltransfer reaction during transcription initiation, occurring in a single crystal, was monitored in real-time by time Raman spectroscopy. Multiple events including substrate binding, conformational changes, metal site formation and disappearance, decrease of substrate and increase of product, are probed individually during the partial or complete reactions occurring on the transcription initiation pathway.
Advisors/Committee Members: Carey, Paul.
Subjects: Biochemistry; Biology; Chemistry; Virology
Keywords: Domain V; group II intron; Raman spectroscopy; RNA; isotope labeling; structure; transcription; RNA polymerase; N4 viron; initiation; NTP; maganesium
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11.
DAI, ZHENYU.
PROTEIN CROSSLINKING BY THE MAILLARD REACTION WITH ASCORBIC ACID AND GLUCOSE.
Degree: PhD, Biochemistry, 2007, Case Western Reserve University
► Nonenzymatic glycation has been implicated in diabetes, aging, and aging related disease…
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▼ Nonenzymatic glycation has been implicated in diabetes, aging, and aging related disease such as Alzheimer’s disease. Nonenzymatic glycation has been implicated in the pathology of normal aging and diabetes. Glycation derived AGEs, especially AGE crosslinks has been hypothesized to be involved in the pathological process. Lens and collagen have been suggested to be major tissues with glycation mediated damage. In lens, ascorbic acid has been believed to be the major glycation agent instead of glucose. No quantitatively important ascorbic acid derived AGE crosslink has been unequivocally elucidated. I therefore hypothesized that the major ascorbic acid derived crosslinks remain to be characterized, which would have potential implication in the pathological process of aging and diabetic lens. On the other hand, glycation mediated crosslinks to collagen have been suggested to be linked with mechanical strength changes of extracellular matrix. I hypothesized that the interfibrillar and intrafibrillar AGE crosslinks will have different impact on mechanical strength of collagen and that detailed information on glycation derived crosslinking sites will be needed to decipher this relationship. As a simplified protein model, RNAse A was used to study the AGE crosslinking sites. In the first part of this dissertation, I isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and D-threose. The chemical structure, a crosslink between lysine and histidine with addition of two threose molecules, was determined by one and two-dimensional NMR spectroscopy combined with LC-tandem mass spectrometry. The compound was found in lens protein incubated with threose at concentrations above 2 mM, but was not detected in the aging human lens. Histidino-threosidine is to our knowledge the first Maillard reaction product known to involve histidine in a crosslink. In the second part of this dissertation, glucosepane, a major AGE crosslink in vivo, was identified following incubation of ribonuclease A with glucose. An intra-molecular glucosepane between K41 and R39 found to be the major crosslink formed in non-oxidative physiological conditions. A minor intra-molecular glucosepane was also observed between K98 and R85. The only inter-crosslink observed between K1 and R39 was an intermolecular DODIC. To our knowledge, this is the first location of intra-molecular and inter-molecular Amadori derived crosslinks in proteins.
Advisors/Committee Members: Monnier, Vincent M.
Subjects: Chemistry, Analytical
Keywords: glycation,; crosslinking,; ascorbic acid,; lens,; collagen,; crosslinking sites,; glucosepane,; mass spectrometry,; NMR,; aging,; diabetes
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12.
Gao, Chengzhuo.
Mechanisms Underlying the Regulation and Functions of HDAC7.
Degree: PhD, Biochemistry, 2008, Case Western Reserve University
► Histone deacetylase 7 (HDAC7), a Class IIa HDAC, is an essential player…
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▼ Histone deacetylase 7 (HDAC7), a Class IIa HDAC, is an essential player in animal development. This protein primarily functions as a transcriptional corepressor of a diverse set of transcription factors. The transcriptional activity of HDAC7 has been shown to be modulated by nucleocytoplasmic shuttling. Nevertheless, the mechanisms underlying the regulation and functions of HDAC7 still remain unclear.CRM1, 14-3-3 proteins, and CaMK play important roles in the trafficking of HDAC7, but the interplay between these proteins in this process is not clearly understood. We show that CRM1 is capable of promoting the cytoplasmic localization of HDAC7 independent of phosphorylation and 14-3-3 binding. Furthermore, our work also identified a novel regulatory mechanism in which HDAC7 can be recruited to distinct subnuclear domains, promyelocytic leukemia protein (PML) nuclear bodies (NBs). We show here that endogenous HDAC7 and PML interact and partially co-localize in PML NBs. Tumor necrosis factor alpha (TNFα) treatment of human umbilical vein endothelial cells (HUVECs) results in the recruitment of HDAC7 to PML NBs and the enhanced association between HDAC7 and PML. We show that by sequestration HDAC7, PML activates HDAC7 target gene, including Matrix Metalloprotease-10 (MMP-10). These results reveal a novel mechanism in which PML blocks the association between HDAC7 and MEF2s by keeping HDAC7 in subnuclear domains. In addition to its role in transcriptional regulation, we explored other potential functions of HDAC7. We show that HDAC7 knockdown reduces the size and the number of the PML NBs in HUVECs. Moreover, HDAC7 stimulates PML sumoylation in vivo and in vitro, suggesting that it possesses a SUMO E3 ligase-like activity. Importantly, HDAC7 knockdown abrogates TNFα-induced PML sumoylation and formation of PML NBs in HUVECs. These results demonstrate a novel function of HDAC7 and provide a regulatory mechanism of PML sumoylation. Lastly, we examined the role of HDAC7 in muscle differentiation. Our work demonstrates that HDAC7 shuttles between the nucleus and the cytoplasm during muscle differentiation. We further demonstrated that constitutive nuclear localization of HDAC7 impairs myogenesis. These results may provide a molecular basis for the development of the potential therapeutic approaches for the treatment of heart and muscular diseases.
Advisors/Committee Members: Kao, Hung-Ying.
Subjects: Biochemistry; Cellular biology; Molecular biology
Keywords: HDAC7; subcellular distribution; transcriptional activity; CRM1; 14-3-3; CaMK; PML NBs; PML sumoylation; SUMO E3 ligase; PML NB formation; Myogenesis
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13.
Geisler, Sarah J.
Decapping of Long Noncoding RNAs Regulates Inducible Genes.
Degree: PhD, Biochemistry, 2012, Case Western Reserve University
► In recent years the complexity of the eukaryotic transcriptome has become a…
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▼ In recent years the complexity of the eukaryotic transcriptome has become a subject of intense curiosity as well as debate. It is now well established, however, in eukaryotic organisms from yeast to humans, that RNA polymerase II (pol II) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs). While our knowledge of the mechanisms and scope of lncRNA-mediated regulation is growing, our understanding of how lncRNAs themselves are regulated is still quite limited. Here I demonstrate that the abundance of many lncRNAs is regulated by decapping-dependent decay. As pol II transcripts, messenger RNAs (mRNAs) and lncRNAs bear distinctive features at their extremities (i.e. a 5’ cap and 3’ poly(A)tail). A highly conserved pathway for the destruction of mRNAs requires removal of the cap structure (i.e. decapping) which then leaves the body of the transcript susceptible to 5’ to 3’ exonucleolytic digestion. Decapping represents a critical control point in regulating mRNA expression because it commits the transcript body to destruction. While the role of decapping in controlling mRNA levels is well documented, the contribution of decapping in modulating the levels and function of other capped RNAs has been largely unexplored. In my study I document that decapping influences the expression of >100 lncRNAs in S. cerevisiae through a novel decapping-dependent pathway that occurs independently of all known mRNA decapping regulators. I find that decapping-sensitive lncRNAs are often expressed proximal to inducible genes. Using galactose inducible genes as a model for lncRNA-mediated regulation, I show that, upon stimulation, clearance of the lncRNA in the nucleus by decapping-dependent decay is required for rapid and robust gene activation. Failure to destabilize this lncRNA, which is known to exert repressive histone modifications, results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. I propose that decappingdependent decay serves a vital role in regulating lncRNA-mediated epigenetic events at inducible genes.
Advisors/Committee Members: Coller, Jeffery.
Subjects: Biochemistry; Biomedical Research; Cellular Biology; Molecular Biology
Keywords: decapping; lncRNA decapping; lncRNA degradation; lncRNA-mediated gene regulation
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14.
Gelfand, Craig Alan.
Structural and functional studies of retroviral nucleocapsid proteins.
Degree: PhD, Biochemistry, 1995, Case Western Reserve University
► The nucleocapsid protein (NC) is an essential, so-called "structural," retroviral protein. NC…
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▼ The nucleocapsid protein (NC) is an essential, so-called "structural," retroviral protein. NC participation is implicated in numerous retroviral processes, including selection, specific dimerization, and histone-like packaging of retroviral genomic RNA; primer RNA annealing; reverse transcription and integration; and virion maturation. This research expands the structural and functional data for two retroviral NCs, aiding in rationalization of their apparent biological complexity. Denaturation studies with avian myeloblastosis virus (AMV) NC reveal approximately 8 kcal/mol of energy stabilizing the native fold of this small protein. Two independent urea-induced structural transitions are detected, by circular dichroism (CD) and intrinsic fluorescence, respectively. These studies suggest that significant structure exists in NCs outside of the highly conserved "cys-his motifs", Cys-X2-Cys-X3-Gly-His-X4-Cys, currently the only regions modeled at atomic resolution. Based on the variety of presumed nucleic acid binding targets, a comprehensive nucleic acid binding study was undertaken to better define and characterize NC participation in the retroviral replication cycle. The nucleic acid affinity of AMV NC, measured by exploiting the anisot ropy of the intrinsic protein fluorescence, is equal (approximately 1 × 104 M-1 in 125 mM NaCl) for an extensive panel of single- and double-stranded RNA and DNA homopolymers (Gelfand et al, 1993. J. Biol. Chem. 268:18450-18456.). This surprising result suggests that all presumed biological roles for NC are indeed possible. Extension of these results toward a universal NC model required subsequent study of the nucleic acid binding properties of another NC. Binding of equine infectious anemia virus (EIAV) NC to single- and double-stranded homopolymeric nucleic acids substantially alters their intrinsic structures. The complex interaction between EIAV NC and nucleic acids, readily apparent by large transitions in the nucleic acid CD spectra, does not induce melting of double stranded structures. The resulting structures might be the basis for the "stimulatory" roles (e.g., enhancing nucleic acid annealing) attributed to NCs. The structural rearrangement of nucleic acids mediated by the lentiviral EIAV NC is not seen with the "oncoviral" AMV NC. This discrepancy represents the first large difference observed between lentiviral and other retroviral structural proteins, and could have significant impact on the search for anti-AIDS drugs
Advisors/Committee Members: Jentoft, Joyce E.
Subjects: Chemistry, Biochemistry
Keywords: Neucleocapsid protein
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15.
Gillaspy, Glenda E.
Expression of genes and differentiation markers in human glioblastoma cell lines.
Degree: PhD, Biochemistry, 1991, Case Western Reserve University
► Glioblastomas are highly malignant tumors found in the central nervous system. To…
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▼ Glioblastomas are highly malignant tumors found in the central nervous system. To define some of their molecular driving forces, transcription rates of oncogenes and growth factors were examined in seven human glioblastoma cell lines and in three normal human glial cell populations. The following genes were over-expressed: PDGF-B in 4/7, EGF-R in 1/7, NEU in 1/7, IGF-2 in 1/7, ROS in 2/7, C-MYC in 2/7, and FOS in 1/7 glioblastomas. PDGF-R was under-expressed in 4/7. This suggests that these genes may be involved in the progression of normal glial cells to the tumor state. No one gene was over-expressed in all glioblastoma cell lines, thus, each had a unique pattern of over-expression. Developmental states of these same cell lines was investigated using antibodies which distinguish glial cell types in the rat optic nerve. The glial origin of each cell line was ascertained using GFAP and 7B11 antibodies. Three cell lines expressed an antigenic phenotype and morphology similar to rat O-2A progenitor cells (A2B5 and HNK-1-positive; GFAP and Gal-C-negative). Two of these cell lines could be forced to undergo differentiation in response to cAMP resulting in type-2 astrocyte-like cells (GFAP-positive) in one case, and in the other case, oligodendrocyte-li ke cells (Gal-C-positive). Three other glioblastoma cell lines appeared to be related to cells from the O-2A lineage, but were heterogeneous and did not undergo identifiable differentiation events following cAMP treatment. One glioblastoma cell line and the normal population were similar to type-1 astrocytes in antigenic phenotype (A2B5 and HNK-1-negative, GFAP-positive) and in morphology. The effect of this in vitro differentiation by cAMP was examined. Morphological alterations were inhibited by serum, and reversible when cAMP was removed. Growth inhibition and increases in glutamine synthetase enzyme activity resulted in 3/4 glioblastoma cell lines treated with cAMP. An overall decrease in the transcription rate of PDGF-B, ROS and IGF-2 resulted in one glioblastoma cell line differentiated with cAMP. PDGF-B mRNA levels were also decreased in this cell line in response to cAMP, but not in another glioblastoma cell line which did not differentiate in the presence of cAMP.
Advisors/Committee Members: Goldthwait, David A.
Keywords: genes human glioblastoma cell lines
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16.
Gong, Bo.
Raman spectroscopic studies of the hepatitis delta virus (HDV) ribozyme.
Degree: PhD, Biochemistry, 2009, Case Western Reserve University
► The general acid/base mechanism proposed for HDV catalysis involves a cytosine, C75…
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▼ The general acid/base mechanism proposed for HDV catalysis involves a cytosine, C75 (genomic) / C76 (antigenomic), in proton transfer. Biochemical studies suggest that C75/C76 has a pKa perturbed to near neutrality. In this thesis, the first objective is to directly measure the pKa of C75 in a genomic HDV ribozyme using Raman crystallography. The results presented here reveal pKa values for C75 that reflect anticooperative thermodynamic coupling with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme. The second objective is to investigate metal-RNA interactions in crystals of HDV ribozyme. Metal cation binding lies at the heart of much of RNA chemistry and is crucial for RNA folding and ribozyme catalysis. Here, we show Raman crystallography and Raman difference spectroscopy provide a unique means for probing metal-RNA interactions in HDV. The present studies focus on: (1) detecting the inner-sphere interactions between magnesium hydrate and the phosphate backbone of HDV by identifying and analyzing the vibrational signatures of inner-sphere coordinated magnesium hydrate (~322 cm-1) and the PO2- symmetric stretch (~ 1100 cm-1) perturbed by the formation of a PO2- —Mg2+ (H2O)x (x≤5) inner-sphere complex. About 5 inner-sphere Mg2+—-O2P contacts per HDV molecule are observed in the presence of 20 mM magnesium; (2) characterizing Mg2+—HDV and Co(NH3)63+—HDV interactions. Our results reveal that Mg2+ and Co(NH3)63+ binding induce modest changes in HDV molecular conformation and Co(NH3)63+ is able to displace some inner-sphere coordinated Mg2+ ions in HDV. Finally, using Raman technique combined with ribozyme kinetic studies (the latter contributions are from the research group of Dr. B.Golden (Purdue University)), we observe an inner-sphere interaction of the catalytic Mg2+ ion with the N7 of guanine in the G1U wobble pair at the cleavage site. A new structural model for the active site of HDV is proposed based on the present results and previous studies. This model accommodates all available biochemical data on the HDV ribozyme.
Advisors/Committee Members: Carey, Paul.
Subjects: Biochemistry
Keywords: Raman spectroscopy; HDV; ribozyme; pKa measurement; Metal ions
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17.
Guo, Canhui.
Mechanism of Anti-Cancer Activity of 9-Aminoacridine Based Drugs.
Degree: PhD, Biochemistry, 2008, Case Western Reserve University
► In the tumors that maintain wild type p53 tumor suppressor gene, p53…
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▼ In the tumors that maintain wild type p53 tumor suppressor gene, p53 activity is frequently inactivated by a variety of mechanisms, such as, overexpression of Mdm2 or MdmX, loss of Arf, or expression of human papilloma virus protein E6. None of these mechanisms, however, explain suppressed p53 function in the majority of renal cell carcinomas (RCC), tumors that largely have wild type p53. In our previous study, we found that a small molecule, 9-aminoacrindine (9AA) can restore p53 function in RCC and strongly stimulate activity of wild type p53 in other types of cancer cells. In addition, these compounds were found capable of inhibiting NF-kB signaling, the pathway that is constitutively active in RCC as well as in the majority of other tumor types. To determine the mechanism of simultaneous modulation of p53 and NF-kB by 9AA we compared global gene and protein expression profiles of two 9AA-treated and untreated RCC cell lines. The set of genes that changed their expression in response to 9AA treatment contained many known p53- and NF-kB targets and therefore was consistent with p53 activation and NF-kB suppression by this compound. Proteomics analysis revealed P110gamma, one of the catalytic subunits of PI3K, to be one of a few proteins that were strongly down-regulated by 9AA treatment. We further found that the observed decrease in P110gamma protein is mediated through the 9AA-induced inhibition of the PI3K/AKT/mTOR signaling pathway. Consistent with the known role of PI3K in regulation of p53 and NF-kB function, knocking down of P110gamma expression by RNA interference in RCC cells inhibited NF-kB and activated p53. Moreover, ectopic overexpression of P110gamma in 293T cells resulted in constitutive activation of NF-kB. Furthermore, overexpression of P110gamma in RCC45 cells made them less sensitive to 9AA treatment indicating, together with all the above-mentioned observations, that p110gamma is one of the targets of 9AA that mediate its modulation of p53 and NF-kB pathways in tumor cells.
Advisors/Committee Members: Gudkov, Andrei.
Subjects: Biochemistry
Keywords: 9-Aminoacridine; p53; NF-kB
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18.
Gurney, Austin Louis.
Characterization of the promoter region of the gene for phosphoenolpyruvate carboxykinase (GTP).
Degree: PhD, Biochemistry, 1992, Case Western Reserve University
► The goal of the research contained within this thesis has been to…
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▼ The goal of the research contained within this thesis has been to identify features of the organization of the PEPCK promoter which enable the appropriate expression of the PEPCK gene. Initial research efforts focused on an examination of the functional significance of the single base difference between the sequence of CRE-1 and CRE-2. This research resulted in the observation that the cAMP responsiveness of the PEPCK promoter could be greatly increased by a single base substitution that altered the CRE-2 sequence to be identical to the CRE-1 sequence. This observation provided early evidence that the mechanism of cAMP regulation could involve the synergistic action of multiple cis elements. Subsequent research pursued identification of specific transcription factors which are able to bind to defined regions of the PEPCK promoter. This work included the discovery that Fos and Jun are able to bind to the PEPCK promoter and acutely modulate the rate of PEPCK transcription. The action of Fos and Jun on PEPCK gene transcription involved each of the Fos/Jun binding sites, and was modulated by additional transcriptional regulatory elements within the promoter. The ability of Fos to inhibit PEPCK transcription was dependent upon P3(I), a region of the promoter which does not bind Fos/Jun heterodimers, but does bind members of the C/EBP family of transcription factors. Stimulation of PEPCK transcription by 8-Br-cAMP or by overexpression of the catalytic subunit of protein kinase A was inhibited by Fos expression. The inhibitory effects of phorbol esters and protein kinase C on PEPCK gene expression may be mediated through the action of Fos and Jun. In addition, several other transcription factors including the thyroid hormone receptor and C/EBPβ have been found to interact with the PEPCK promoter. The identification of these factors and the examination of their ability to alter the rate of transcription has expanded our knowledge of the organization the PEPCK promoter.
Advisors/Committee Members: Hanson, Richard W.
Subjects: Biology, Molecular
Keywords: promoter region; phosphoenolpyruvate carboxykinase; GTP
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19.
Hake, Michael James.
Spectroscopic Characterization of the Interaction of Nck Domains with the Epidermal Growth Factor Receptor Juxtamembrane Domain.
Degree: PhD, Biochemistry, 2008, Case Western Reserve University
► Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2)…
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▼ Nck is a ubiquitously expressed adaptor protein containing Src homology 2 (SH2) and Src homology 3 (SH3) domains. It integrates downstream effector proteins with cell membrane receptors, such as the epidermal growth factor receptor (EGFR). EGFR plays a critical role in cellular proliferation and differentiation. The 45-residue juxtamembrane (JM) domain of EGFR, located between the transmembrane and kinase domains, regulates receptor activation and trafficking to the basolateral membrane of polarized epithelia through a proline-rich motif that resembles a consensus SH3-domain binding site. We demonstrate here that the JM region can bind to Nck, showing a notable binding preference to the second SH3 domain. To elucidate the structural determinants for this interaction, we have solved the NMR solution structures of both the first and second Nck SH3 domains (Nck1-1 and Nck1-2). These domains adopt a canonical SH3 β-barrel-like fold, containing five anti-parallel strands separated by three loop regions and one 310 helical turn. Chemical shift perturbation studies have identified the residues that form the binding cleft of Nck1-2, which are primarily located in the RT and n-Src loops. The JM domain binds to Nck1-2 with an affinity of ~80 μM through a positively-charged sequence near the N-terminus, as opposed to the polyproline sequence. The two Nck SH3 domains exhibit both steric and electrostatic differences in their RT-Src and n-Src loops, and a model of the Nck1-2 domain complexed with JM highlights the factors that define the putative binding mode for this ligand.
Advisors/Committee Members: Sönnichsen, Frank.
Subjects: Biochemistry; Biophysics
Keywords: Protein-protein interaction; signaling; adaptor protein; NMR; structure
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20.
Hakjung, Kim.
Studies on site-specifically modified human dihydrolipoamide dehydrogenase.
Degree: PhD, Biochemistry, 1992, Case Western Reserve University
► The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase…
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▼ The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in E. coli using a λ P L promoter-driven prokaryotic expression vector. Since wild-type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli JRG1342. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 possessed an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about 36% of the mature E3 activity. The double reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. This study indicated that the mature recombinant human E3 was an active enzyme whose kinetic properties were similar to those of mammalian E3s. Two site-specifically mutated human E3s (His-452 → Gln and Glu-457 → Gln) were expressed in E. coli JRG1342. The double reciprocal plot for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its K m for dihydrolipoamide was dramatically increased by about 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-452 mutant E3 possessed about 2-fold higher activity at pH 8.5 than the activity at pH 8.0, indicating that the Gln-452 mutant E3 probably catalyzed the reaction with the help of water molecules (hydroxyl ions) in assay solution. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed the E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on a double reciprocal plot. Its specific activity decreased to 28% of that of wild-type enzyme. Its K m for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in E3 reaction, is critical to E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.
Advisors/Committee Members: Patel, Mulchand S.
Subjects: Chemistry, Biochemistry
Keywords: modified human dihydrolipoamide dehydrogenase
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21.
Hejlik, Daniel Paul.
Investigations of the pyruvate binding site in the 5S subunit of transcarboxylase.
Degree: PhD, Biochemistry, 1995, Case Western Reserve University
► Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme that catalyzes the reversible…
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▼ Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme that catalyzes the reversible transfer of a carboxyl group from methylmalonyl CoA to pyruvate, forming propionyl CoA and oxaloacetate. The enzyme is composed of a central 12S hexamer to which six 6S complexes are attached. Each 6S complex contains a 5S dimer and two 1.3S monomers. This work is concerned with the characterization of the pyruvate binding sites present in the 5S dimer. The initial work was concerned with solving previous problems with the stable expression of 5S as an active dimer. Coexpression of 5S and 1.3S in the same bacterial cells failed to result in the formation of the more stable 6S complex. The pET system was used for the overexpression of 5S but most of the protein obtained was insoluble. A purification protocol was developed with the previously constructed 5S expression system that allowed for the purification of sufficient quantities of active 5S dimer. Diethyl pyrocarbonate (DEPC) modification of histidine residues in 6S purified from P. shermanii indicated that histidine residues were necessary for the function of 6S in the transcarboxylase reaction. Preincubation with the substrate pyruvate prevented inactivation. It was revealed that pyruva te prevented the modification of one histidine residue per pyruvate binding site. A decrease in the Vmax of the transcarboxylase reaction suggested that this residue was involved in catalysis. Pyruvate binding and transcarboxylase assembly were not affected by the modification. Preliminary peptide mapping experiments suggested that the protected residue was His-25 of 5S. Photooxidation of histidine residues in 6S with rose bengal also resulted in the inactivation of 6S. However, unlike the results with DEPC, preincubation with pyruvate did not prevent inactivation. Pyrene glyoxal modification of arginine residues and iodoacetate modification of cysteine residues in 6S resulted in different degrees of inactivation. Preincubation with pyruvate failed to prevent the inactivation in both cases. A PCR mutagenesis protocol was developed for 5S and used to mutate Trp-73 to alanine. This system is versatile and can be adapted for mutagenesis of other residues in 5S.
Advisors/Committee Members: Samols, David.
Keywords: pyruvate binding site; 5S subunit; transcarboxylase
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22.
Huang, Kristen Marie.
Protein glycosylation studies in mammalian cells and yeast.
Degree: PhD, Biochemistry, 1994, Case Western Reserve University
► Protein glycosylation occurs within the endoplasmic reticulum and the Golgi complex. In…
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▼ Protein glycosylation occurs within the endoplasmic reticulum and the Golgi complex. In mammalian cells, cell surface membrane glycoproteins become glycosylated as they move through the rough endoplasmic reticulum. The oligosaccharide chains are processed as the glycoprotein moves through the Golgi with the terminal sugar residues, galactose and sialic acid added in the trans-Golgi cisternae and trans Golgi network respectively. Following glycosylation membrane glycoproteins are transported to the cell surface. We have investigated the return of membrane glycoproteins to the Golgi complex after their appearance on the cell surface. We have found that, in a Chinese hamster ovary cell line, the mannose 6-phosphate receptor returns to the galactosyltransferase compartment of the Golgi complex. In addition, at least ten other cell surface glycoproteins follow this recycling pathway with similar kinetics. By analyzing a subset of membrane glycoproteins, we have determined that a significant fraction of the glycoprotein flux through the Golgi is attributable to recycling cell surface glycoproteins. Many of the steps in glycoprotein synthesis are conserved between mammalian cells and yeast cells. To better understand the process of glycosylation, we have isolated mutants in the yeast Schizosa ccharomyces pombe that are defective in protein glycosylation. These mutants synthesize and secrete glycoproteins that have smaller molecular weights than wild type strains. An analysis of the glycoprotein oligosaccharide chains revealed two of the mutants are defective in core oligosaccharide modification. One of these mutants is defective in UDP glucose 4-epimerase activity which results in decreased levels of UDP galactose and undergalacosylation of cellular glycoproteins. The other mutant is more likely defective in a glycosyltransferase. The isolation of such mutants will aid in cloning Schizosaccharomyces pombe genes involved in protein glycosylation.
Advisors/Committee Members: Snider, Martin.
Keywords: Protein glycosylation studies mammalian cell yeast
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23.
Hu, Wenqian.
The Interplay of Eukaryotic mRNA Translation and Degradation.
Degree: PhD, Biochemistry, 2010, Case Western Reserve University
► The regulation of mRNA translation and degradation is essential for proper gene…
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▼ The regulation of mRNA translation and degradation is essential for proper gene expression. In eukaryotes, a major mRNA degradation pathway is initiated by deadenylation, followed by decapping, and ultimately 5’-3’ exonucleolytic digestion. Removal of the mRNA 5’ cap is an irreversible step in mRNA degradation, and is postulated to require dissociation of the mRNA from the ribosomes and packaging into sub-cellular, ribosome-free granules termed P-bodies. Based on this and other observations, a “two-step” model of eukaryotic mRNA degradation had been proposed that mRNA translation and degradation occur in different compartments within the cell. Recent findings suggest, however, that mRNA degradation may occur independent of P-bodies. Consequently, an important but unresolved issue in the field is to determine the context in which mRNA is degraded. In my study, I have demonstrated that the three steps of mRNA decay, deadenylation, decapping, and 5’-3’ exonucleolytic digestion, occur co-translationally. Specifically, mRNA deadenylation does not lead to ribosome dissociation. Second, I found that decapped mRNA mainly bound to polyribosomes, suggesting that decapping occurs during translation. In addition, using kinetic analysis, I demonstrated that mRNA decapping is initiated while the mRNA is on polyribosomes. Third, I observed that in wild-type cells, exonucleolytic decay fragments are polyribosome associated when ribosome transit is slowed in cis. Lastly, using an innovative assay I designed, I detected decapping products from endogenous mRNAs mainly on polyribosomes in wild type cells. These results clearly demonstrate that under normal physiological conditions, mRNA degradation occurs while the mRNA is still associated with ribosomes. In addition to the normal mRNA turnover pathway, I observed that mRNA decapping triggered by nonsense-mediated mRNA decay, an important mRNA quality control mechanism, also occurs on polyribosomes. Collectively, these results indicate that polyribosomes are the major sites for destroying both normal and aberrant mRNAs within eukaryotic cells
Advisors/Committee Members: Coller, Jeff.
Subjects: Biochemistry; Cellular biology; Genetics; Molecular biology
Keywords: mRNA turnover, mRNA decapping, mRNA translation, polyribosomes
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24.
Jacques, Pamela Susan.
In vitro reconstitution as a strategy for evaluating the subunits of human immunodeficiency virus type 1 reverse transcriptase.
Degree: PhD, Biochemistry, 1995, Case Western Reserve University
► Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for…
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▼ Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for viral replication and possesses DNA-and RNA-dependent DNA polymerase, ribonuclease H (RNase H), and RNase H* (hydrolysis of double stranded RNA) activities. The heterodimer (p66/p51) is the biologically relevant and the enzymatically active form of RT which initiates minus strand DNA synthesis from the HIV-1 replication primer, human tRNA Lys,3. Any of the above enzymatic functions, including dimerization, could possibly be inhibited in an effort to inactivate the enzyme and therefore be used to develop therapeutic strategies for slowing the progression of AIDS. In vitro reconstitution and mutagenesis of p66 and p51 HIV-1 RT were utilized in an attempt to localize specific activities within these subunits. Three truncated HIV-1 51 kDa subunits were constructed containing C-terminal deletions of 13, 19 and 25 amino acids. These p51 deletion mutants were then reconstituted with wild type HIV-1 p66 in an effort to determine a functional role of p51 in the context of the biologically significant heterodimer. Deletion of the C-terminal 25 residues of p51 resulted in the inability to form a stable heterodimer. However, deletion of 13 or 19 amino acids did not disrupt dimerization, but significantly affected the various activities of heterodimer HIV-1 RT. Our study indicates the C-terminal region of p51 is necessary for retention of both tRNA Lys,3 affinity and RNase H activity. In a separate study, p66 HIV-1 mutants containing single amino acid substitutions between residues Glu224 and His235 were constructed. These altered 66kDa subunits were then reconstituted with wild-type p51 to generate p66 E224A/p51-p66 E233A/p51 and p66 H235A/p51 (reconstitution of p66 L234A and p51 did not result in a stable heterodimer). Each selectively-mutated heterodimer was assayed for retention of DNA- and RNA-dependent DNA polymerase activities, RNase H activity, and template-primer and tRNA Lys,3 primer binding. Our data indicates p66 residues Trp229-Tyr232 are essential for both DNA polymerase activities and template-primer affinity
Advisors/Committee Members: Le Grice, Stuart F. J.
Subjects: Chemistry, Biochemistry
Keywords: HIV-1; In vitro reconstitution
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25.
Jafarifar, Faegheh.
IDENTIFICATION OF CARE-BINDING MICRORNAS TARGETING VEGFA AND THE ROLE OF HNRNP L IN MODULATION OF THEIR ACTIVITIES.
Degree: PhD, Biochemistry, 2011, Case Western Reserve University
► Expression of vascular endothelial growth factor A (VEGFA) by tumor-associated macrophages (TAMs)…
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▼ Expression of vascular endothelial growth factor A (VEGFA) by tumor-associated macrophages (TAMs) is critical for tumor progression and metastasis. Hypoxic microenvironment of solid tumors induces VEGFA expression in TAMs by increased transcription and mRNA stabilization. VEGFA mRNA stabilization mediated by RNA binding proteins (RBPs) such as heterogeneous nuclear ribonucleoprotein L (hnRNP L) plays a major role in VEGFA regulation. hnRNP L binds to a specific CA-rich element (CARE) in the VEGFA 3’-untranslated region (3’-UTR). Our laboratory has identified an RNA conformational switch in human VEGFA 3’-UTR that integrates signals from interferon gamma (IFN-γ) and hypoxia to regulate VEGFA expression in human macrophage-like monocytic cells. The RNA conformational change in adjoining elements GAIT (IFN-γ-activated inhibitor of translation) and CARE is dictated by mutually exclusive, stimulus-dependent binding of proteins. These proteins include the heterotetrameric IFN γ-activated inhibitor of translation complex (GAIT) and hnRNP L. This dissertation is focused on the interplay between microRNAs (miRNAs) and RBP that regulates the expression of one of the most important angiogenic factors VEGFA. We identified a new mechanism of stress-dependent VEGFA regulation that involves reversal of miRNA-mediated silencing of VEGFA expression by hnRNP L. The data indicate that the CARE element of human VEGFA 3’-UTR, the binding site of hnRNP L, is targeted by miRNAs. Among these miRNAs, miR-297 and -299 are endogenously expressed in monocytic cells and negatively regulate VEGFA expression. Our data revealed that the repressive effect of these miRNAs can be inhibited in hypoxia. We showed the direct role of hnRNP L and established a mechanism for this observation. In hypoxia, the hnRNP L expression does not change but rather its cellular localization does. Hypoxia induces translocation of nuclear hnRNP L to the cytoplasm in a VEGFA mRNA independent manner and markedly increases cytoplasmic hnRNP L concentration which competes away the CARE-binding miRNAs. These studies introduce a novel mechanism of VEGFA gene regulation and can contribute to development of miRNA-based anticancer therapeutic tools.
Advisors/Committee Members: Fox, Paul.
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26.
Jaladat, Yasaman.
Identification and characterization of U6 and U2 small nuclear RNAs as the key players of the splicing reaction: Introducing a minimal RNP system.
Degree: PhD, Biochemistry, 2011, Case Western Reserve University
► Pre-mRNAs in higher eukaryotes undergo a complicated process called splicing to remove…
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▼ Pre-mRNAs in higher eukaryotes undergo a complicated process called splicing to remove the introns and ligate the exons. This is a crucial step in eukaryotic gene expression, which is processed by the spliceosome, a highly complex ribonucleoprotein. Many fundamental aspects of the spliceosome are still unknown. Although the base paired complex of U6 and U2 snRNAs, in the absence of ~150 spliceosomal proteins, catalyzes the first step of splicing in vitro, it is critical to further develop this model system to unravel the structural and functional properties of the spliceosome. First, we show that the U6 and U2 complex is competent to perform a two-step reaction, which is chemically identical to the spliceosomal splicing reaction and is dependent on and occurs in proximity of sequences that are known to be critical for splicing in vivo (ACAGAGA sequence). Furthermore, we analyze the U6 and U2-catalyzed reaction and show that while the 5′ and 3′ splice sites are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. This snRNA-catalyzed reaction is metal ion-dependent and terbium(III) cleavage reactions indicate metal binding by the U6 and U2 complex within the evolutionarily conserved regions of U6. We further demonstrate that the U6 and U2 complex can perform splicing in cis by removal of an intervening sequence from a model substrate. Next, we observe that addition of the C terminal domain of Prp8 (CTD fragment), a highly conserved important spliceosomal protein, can enhance the rate of the splicing reaction in vitro in a specific manner and the wild type sequence and the intact folded structure of the CTD fragment are necessary for the rate enhancement. We determine that the level of enhancement is dependent on the concentration of the CTD fragment, which is able to physically interact with U6 and U2. Together, our observations support a model in which the the CTD fragment of Prp8 possesses a chaperone-like activity to enhance splicing.
Advisors/Committee Members: Merrcik, William.
Subjects: Biochemistry
Keywords: Invitro splicing reaction; U6 and U2 snRNAs; Prp8
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27.
Jia, Huijue.
Molecular Mechanism of the TRAMP Complex.
Degree: PhD, Biochemistry, 2011, Case Western Reserve University
► The TRAMP complex (Trf4/Air2/Mtr4 polyadenylation) is required for quality control and 3’…
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▼ The TRAMP complex (Trf4/Air2/Mtr4 polyadenylation) is required for quality control and 3’ maturation of many RNAs in the nucleus. The TRAMP complex consists of three units, the poly(A) polymerase Trf4/5p, the zinc-knuckle protein Air1/2p, and the Ski2-like RNA helicase Mtr4p. Using recombinant Saccharomyces cerevisiae TRAMP complex, I found that the RNA helicase Mtr4p modulates activities of the polymerase Trf4p and restricts the length of oligo(A) appended after addition of ~4 nt. The modulation does not require duplex unwinding but relies on sensing of oligo(A) length by Mtr4p. Trf4p, in turn, stimulates the unwinding activity of Mtr4p independent of polyadenylation. Polyadenylation of duplex RNAs by Trf4p creates a landing site for Mtr4p to start unwinding, the length of oligo(A) required coincides with restriction of adenylation by Mtr4p. The functional cross-talk between Trf4p and Mtr4p in the TRAMP complex promotes addition of a minimum oligo(A) tail (~4 nt) and may be critical for sorting of polyadenylated RNAs in vivo and for subsequent specific target processing by the nuclear exosome. The S. cerevisiae DEAD-box protein Ded1p unwinds RNA duplexes by local strand separation instead of translocation. In this study, I established that distinct units of Ded1p exist on single-stranded and duplex regions; Ded1p unit(s) bound to single-stranded regions facilitates loading of additional unit(s) of Ded1p to duplex regions for unwinding. I further analyzed Ded1p binding to single-stranded RNA in the presence of analogs for different states of ATP hydrolysis. Each Ded1p monomer binds ~10 nt ssRNA in the presence of the non-hydrolyzable analog ADPNP. The ground state analog ADP-BeFx and the transition state analog ADP-AlF4 lead to binding of more Ded1p molecules to the same length of RNA, probably through Ded1p oligomerization. Such ATP analog-induced association of additional Ded1p molecules likely takes place during duplex unwinding.
Advisors/Committee Members: Jankowsky, Eckhard.
Subjects: Biochemistry
Keywords: RNA helicase; RNA duplex unwinding; poly(A) polymerase; polyadenylation; TRAMP; Mtr4; Ded1; ATP analog
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28.
Jin, Ming-Jie.
Intracellular transport pathway of cell surface receptors to the Golgi complex.
Degree: PhD, Biochemistry, 1990, Case Western Reserve University
► Membrane traffic between endocytic and exocytic organelles has recently been established. Some…
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▼ Membrane traffic between endocytic and exocytic organelles has recently been established. Some cell surface receptors are transported to the Golgi complex. In this dissertation, I used cation-independent mannose 6-phosphate receptor (MPR CI) and transferrin receptor (TfR) as model molecules to study and characterize the cell surface to the Golgi transport pathway. MPR CI functions in the packaging of both newly-made and extracellular lysosomal enzymes into lysosomes. To learn about the intracellular pathway followed by surface receptor, we examined the entry of surface MPR CI into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 human leukemia cells. When the cells were recultured at 37°C, surface MPR CI was resialylated with a half-time of 1-2 h. Resialylation was inhibited by reduced temperature, a treatment that allows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPR CI is transported to the sialyltransferase compartment in the Golgi complex. After resialylation, 10-20% of the receptor was found on the cell surface, suggesting that surface MPR CI mixes with the intracellular pools after endocytosis. TfR follows complex intracellu lar pathways. While most receptors are cycling between the cell surface and endosomes, a small fraction is transported to the Golgi complex. In order to examine the role of microtubules in these transport processes and to characterize the surface-to-Golgi pathway, we studied TfR dynamics in K562 cells treated with nocodazole to depolymerize microtubules. Nocodazole treatment caused a decrease in the endocytosis rate of surface TfR without affecting the exocytosis rate of internalized receptor to the cell surface. This resulted in a 50% increase in the steady state level of surface TfR. However, these changes did not have significant effects on the overall rate of TfR cycling. Nocodazole did not affect transport of TfR from the cell surface to the Golgi complex, demonstrating that microtubules are not required for this transport process. Moreover, nocodazole was found to interfere with transport from endosomes to lysosomes, which was demonstrated by following the fluid-phase marker horseradish peroxidase. Thus, the cell surface-to-Golgi pathway does not involve a lysosomal intermediate.
Advisors/Committee Members: n/a, n/a.
Subjects: Chemistry, Biochemistry
Keywords: cell surface receptors Golgi complex
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29.
Kalp, Matthew Douglas.
Raman Crystallographic Studies of Inhibitor Reactions in Class A β-Lactamases.
Degree: PhD, Biochemistry, 2009, Case Western Reserve University
► Raman spectroscopy involves analyzing inelastically scattered photons from a laser beam that…
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▼ Raman spectroscopy involves analyzing inelastically scattered photons from a laser beam that is focused into the sample of interest. The laser light interacts with the sample, which results in the energy of the laser photons being shifted up or down. The shift in energy—the Raman spectrum—provides information on molecular vibrations that, in turn, yield data on molecular conformation, electronic distribution, and environment. At its most effective, Raman spectroscopy can provide exquisite detail from an important site or molecule in a much larger macromolecular complex. In this work, Raman crystallography is used to described the reaction pathway between small molecule inhibitors and class A β-lactamases. β-lactamases are enzymes (EC 3.5.2.6) produced by both Gram-negative and Gram-positive bacteria and are responsible for their resistance to β-lactam antibiotics, such as penicillins, cephalosporins, and carbapenems. These antibiotics have a common ring structure, known as a β-lactam, which consists of three carbon atoms and one nitrogen atom. The lactamase enzymes are hydrolases that break the ring open, deactivating the molecule's antibacterial properties. Substrates, such as penicillins and 1st and 2nd generation cephalosporins, are rapidly hydrolyze by the majority of β-lactamases. Only inhibitors, such as penam sulfones, form a transiently stable acyl-enzyme with the β-lactamase, which effectively prevents the enzyme from hydrolyzing other β-lactams. Most inhibitors—penam sulfones, clavams, carbapenems, and cephalosporins—undergo secondary reactions inside the enzyme. This results in multiple reaction intermediates and, despite intense research efforts, it is still unclear which species is responsible for the clinical utility of these compounds. This work will describe the reaction pathways for various inhibitors with SHV β-lactamase and its variants by Raman difference spectroscopy in single protein crystals. In this method, one measures an enzyme spectrum and the spectrum of an enzyme complexed with ligand. Subtraction of the two yields the spectrum of the bound ligand and any conformation changes in the protein that occur upon complexation. With the ability to probe unique molecular structures and conformations, Raman crystallography is ideal for identifying and observing populations and kinetic behavior of reaction intermediates and intermediates constituting less than 30% active site occupancy.
Advisors/Committee Members: Anderson, Vernon.
Subjects: Biochemistry; Biophysics
Keywords: SHV-1; Beta-lactamase; tazobactam; sulbactam; clavulanate; clavulanic acid; mechanism-based inhibitors; Raman spectroscopy; Raman crystallography
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30.
Kang, Li.
Chronic Ethanol Feeding Disrupts Both Lipid and Glucose Homeostasis in Rat Adipose Tissue.
Degree: PhD, Biochemistry, 2007, Case Western Reserve University
► Chronic ethanol consumption induces hepatic steatosis and represents an independent risk factor…
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▼ Chronic ethanol consumption induces hepatic steatosis and represents an independent risk factor for type 2 diabetes, associated with the disruption of lipid and glucose homeostasis. Adipose tissue acts as a regulator for maintaining whole-body lipid and glucose homeostasis; therefore, it was hypothesized that lipid and glucose homeostasis in adipose tissue might be vulnerable to chronic ethanol exposure. Increases in lipolysis, and the resulting release of free fatty acids into the circulation, have been associated with the development of insulin resistance and liver injury in other model systems. Therefore, the effects of chronic ethanol feeding on regulation of triglyceride metabolism in adipose tissue were first investigated. The current study has demonstrated that chronic ethanol feeding to rats increased the turnover rate of triglycerides in epididymal adipose tissue; the rate of triglyceride synthesis was increased 1.9-fold and the rate of triglyceride degradation was increased 3.1-fold over pair-fed controls. Triglyceride degradation is stimulated by b-adrenergic receptor activation and inhibited by insulin. Since chronic ethanol increased the rate of triglyceride degradation, it was first hypothesized that the lipolytic responses of adipocytes to b-adrenergic receptors was increased by chronic ethanol. However, chronic ethanol feeding actually suppressed b-adrenergic receptor-stimulated lipolysis, both in vivo and ex vivo. This suppression of b-adrenergic receptor-stimulated lipolysis by chronic ethanol was associated with inhibited intracellular cAMP accumulation and coincident repression of cAMP-dependent PKA activation and phosphorylation of perilipin A and HSL. These data suggest that increased stimulation of lipolysis by b-adrenergic receptors did not contribute to increased triglyceride degradation after chronic ethanol feeding. In contrast to the role of b-adrenergic agonist in the activation of lipolysis, insulin is the key inhibitor of lipolysis. The effect of chronic ethanol on the anti-lipolytic action of insulin was then studied. Chronic ethanol feeding markedly impaired insulin-mediated suppression of lipolysis in adipocytes isolated from epididymal adipose tissue, as well as in conscious rats, during a hyperinsulinemic-euglycemic clamp. Taken together, these data indicate that chronic ethanol feeding increased the rate of triglyceride degradation due to a loss in the anti-lipolytic response of adipocytes to insulin. The effects of chronic ethanol consumption on glucose homeostasis in adipose tissue were also investigated utilizing the hyperinsulinemic-euglycemic clamp technique. Hyperinsulinemic-euglycemic studies revealed that chronic ethanol feeding to rats decreased whole-body glucose utilization and impaired the ability of insulin to inhibit hepatic glucose production. Since adipose tissue and skeletal muscle are the two major organs utilizing glucose in response to insulin, the relative contribution of these two tissues in mediating impaired glucose utilization after chronic ethanol feeding was further determined. While glucose disposal in skeletal muscle did not differ between pair- and ethanol-fed rats, glucose disposal in adipose tissues, including epididymal, subcutaneous, and omental depots, was decreased in ethanol-fed rats compared to pair-fed rats during the hyperinsulinemic-euglycemic clamp. These data demonstrate that chronic ethanol feeding decreased whole-body glucose utilization by impairing the utilization of glucose by adipose tissue, rather than skeletal muscle. In summary, this thesis has demonstrated that chronic ethanol feeding disrupted both lipid and glucose homeostasis in adipose tissue.
Advisors/Committee Members: Nagy, Laura.
Keywords: ETHANOL; CHRONIC ETHANOL FEEDING; lipolysis; ADIPOSE TISSUE
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