The antibody class switch is achieved by DNA recombination between the sequences called switch (S) regions located 5' to immunoglobulin (Ig) heavy chain constant (CH) region genes. This process can be induced in cultured B cells by polyclonal stimulation and switching can be directed to specific antibody classes by certain lymphokines. These stimuli may regulate the accessibility of CH genes and their S regions to a recombinase as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged (germ-line) CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germ-line CH genes is unknown. How stimuli regulate the accessibility of CHgenes is also unclear. I report in this dissertation the structure of the RNA transcribed from the unrearranged Cγ1 gene in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4 (rIL-4). I will also show that an 150-bp region upstream of the first initiation site of germ-line γ1 RNA contains promoter and enhancer elements responsible for basal level expression and inducibility by phorbol 12-myristate 13-acetate (PMA) and synergy with IL-4 in an IgM+ B cell line, L10A6.2, and an IgG2a+B cell line, A20.3. The germ-line γ1 RNA is initiated at multiple start sites 5' to the tandem repeats of the γ1 switch (Sγ1) region. As is true for analogous RNAs transcribed from other unrearranged genes, the germ-line γ1 RNA has an I exon transcribed from the region 5' to the Sγ1 region.. The Iγ1 exon is spliced at a unique site to the Cγ1 gene. The germ-line γ1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acids in length. Elements located within the 150 bp region 5' to the first initiation site of germ-line γ1 RNA are necessary and sufficient to confer inducibility by PMA and synergy with IL-4 to a minimal thymidine kinase (TK) promoter in L10A6.2 cells but are not sufficient to confer this inducibility in A20.3 cells. Linker-scanning mutations demonstrated that these multiple elements function in a mutually dependent manner as indicated by the fact that mutation of any single element will decrease constitutive expression and inducibility by PMA and PMA plus IL-4. This 150-bp region contains several consensus sequences that bind to known or putative transcription factors, including a C/EBP binding site/IL-4 response element (in the promoter for Ia Aαkgene), four CACCC boxes, a PU box, a TGFβ inhibitory element (TIE), an interferon-αβ response element (αβIRE), and an AP-3 site. My results begin to provide a description of the mechanism of regulation of the accessibility of unrearranged germ-line Sγ1-Cγ1 gene. By activating the germ-line γ1 promoter, IL-4 induces transcription of germ-line γ1 RNA, thereby inducing accessibility of the Sγ1-Cγ1 gene. By inhibiting expression of the germ-line γ1 promoter, IFNγ and TGFβ down-regulate transcription of germ-line γ1 RNA, thus reducing the accessibility of the Sγ1-Cγ1 gene. My results also suggest that signaling via the antigen receptor on B cells may be involved in induction of switch to IgG1. Furthermore, this is the first case reported in which multiple functionally interdependent elements are needed to respond to PMA.

The steroid/thyroid hormone receptor superfamily is a large class of ligand-dependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development, homeostasis, and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basal level via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors, particularly the vitamin D, estrogen, and retinoid receptors, and modulates their transcriptional activity. It was discovered that these receptors interact with different α-helical LXXLL motifs of RAC3 in vitro. Mutation of specific motifs differentially impairs the ability of RAC3 to enhance transcription by the receptors in vivo. In addition, the intrinsic transcriptional activation function of RAC3 was also characterized. Here, a single LXXLL motif, NR box v, was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent, the coactivator pocket of RARα overlaps with the corepressor pocket, with the exception of helix 12, which is required for coactivator, but not corepressor binding. Recruitment of RAC3 or SMRT also correlates directly with the ability of RARα to activate or repress transcription, respectively. Intriguingly, it was discovered that the AF-2 domain of RXRα inhibited cofactor binding to RXRα heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXRα cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXRα is the primary docking point for cofactors at RXRα heterodimeric complexes. Taken together, this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.

CD8 T cells recognize complexes of MHC class I and peptide on the surface of target cells. MHC class I antigen presentation is a long pathway, in which proteins are degraded by proteasomes to generating oligopeptides, which may be further trimmed by aminopeptidases in the cytosol. Peptides are transported into the ER, where they may be further trimmed by ER lumenal aminopeptidases and bind to newly-synthesized MHC class I complexes. Proteins degraded by the proteasome are generally tagged with ubiquitin by a combination of ubiquitin-conjugating enzymes and ubiquitin ligases. UBC9 is one ubiquitin conjugating enzyme, which does not conjugate ubiquitin, but instead conjugates small ubiquitin-like molecules (SUMO) to target protein. UBC9 has been found to regulate the functions of many proteins in vivo, most importantly by modifying nuclear transportation and function. Curing [During] my thesis work, I studied the function of UBC9 in MHC class I antigen presentation. UBC9 over-expression in COS cells co-expressing ovalbumin markedly increased presentation SIINFEKL (the immunodominant epitope from ovalbumin in the context of H-2Kb), and UBC9 overexpression increased cell surface H-2Kbin general, suggesting that Ubc9 increased MHC class I antigen presentation by increasing peptide supply. UBC9 did not increase synthesis or degradation of ovalbumin. In transient transfection experiments, Ubc9 increased presentation of SIINFEKL precursors that did, and that did not, depend on proteasomes for processing, as well as SIINFEKL precursors targeted to the ER, bypassing cytosolic processing altogether. However, a C-terminal extended precursor of SIINFEKL, which requires only proteasomal processing before presentation, was the most markedly affected by UBC9 overexpression. This suggested that UBC9 was affecting the pattern of cleavages made by proteasomes in ways that enhance the generation of the C-terminus of SIINFEKL. Because presentation of SIINFEKL itself (which requires no further proteolytic processing) was also enhanced, UBC9 must also affect steps in the class I pathway that occur after the generation of the mature epitopes. UBC9 did not affect the rate of peptide degradation in cytosolic extracts or in intact cells. These findings suggested that UBC9 might have multiple effects on the MHC class I antigen presentation pathway. Immunofluorescent microscopy demonstrated that UBC9 increased the expression of the beta subunits of immunoproteasomes (LMP2, LMP7, and MECL1) as well as of TAP1 and tapasin. In contrast, UBC9 expression did not increase levels of calnexin, calreticulin, ERp57, or Protein disulfide isomerase (PDI). Similarly, levels of leucine aminopeptidase were not increased in UBC9-transfected cells. Therefore, UBC9 overexpression increases the levels of some but not all components of the class I pathway. UBC9 overexpression increased protein levels of MECL1, LMP2 or LMP7 that were under the control of viral promoters, and levels of MECL1 mRNA were similar in control vector and UBC9 transfected cells. Therefore, UBC9 did not increase the level of expression of these subunits through increased transcription. Pulse-chase experiments showed that UBC9 overexpression reduced the degradation of MECL1. Therefore, UBC9 increases the levels of at least some of these components of the MHC class I antigen presentation pathway by increasing their stability. To know the biological significance of UBC9 in MHC class I antigen presentation, I used small interfering RNA (siRNA) to knock down UBC9. Though UBC9 can be successfully knocked down by siRNA, the UBC9-negative cells became very sick, and were not suitable for the study of MHC class I antigen presentation. There are three forms of SUMO molecules in mammalian cells: SUMO-1, SUMO-2 and SUMO-3. My study suggested that SUMO-2 may be involved in UBC9's regulation of MHC class I antigen presentation, since mutant SUMO-2 blocked UBC9's ability to increase H-2Kb-SIINFEKL levels on the cell surface after the cells were loaded with ovalbumin. To further study the function of UBC9, I mutated the active amino acid Cys 93 of UBC9 to Ser (UBC9OH). Unexpectedly, this mutant form (UBC9OH) has very similar effects as wild-type UBC9, increasing Kb-SIINFEKL levels at the cells surface. This suggested that UBC9 protein regulates MHC class I antigen presentation pathway proteins by direct or indirect protein interaction, rather than (or as well as) by SUMO conjugation. Taking account of SUMO-2 results, I propose that wild-type UBC9 (either transfected or endogenous) conjugates SUMO-2 to its substrates, and then UBC9 (wild-type or mutant) interacts with its sumoylated targets, thus affecting protein functions. I also studied heat shock protein Hsp27, which is known to be a substrate for UBC9 in vivo. Hsp27 is expressed in a variety of tissues in the absence of stress, and may regulate actin dynamics. Hsp27 overexpression decreased generation of H-2Kb-SIINFEKL complexes from SIINFEKL precursors that did, and did not, require proteasomes for processing, or that were targeted to the ER. Hsp27 over-expression did not affect protein synthesis, and globally decreased cell surface H2-Kb and H2-Dblevels, but did not affect HLA-A0302 level. Hsp27 overexpression inhibits the presentation of ER-localized SIINFEKL. Taken together, my data suggested that HSP27 may inhibit MHC class I antigen presentation by affecting MHC class I molecules itself rather than peptide supply. After Hsp27 was eliminated with siRNA, the effects were very similar to those seen with Hsp27 overexpression. Levels of H-2Kb-SIINFEKL decreased, and overall cell surface H-2Kb and H-2Db levels decreased. It is possible that when Hsp27 is over-expressed, it acts as a dominant negative form, conferring a similar phenotype to Hsp27 knockdown. These observations suggest that Hsp27 plays an important role in MHC class I antigen presentation.

The synapse is the primary locus of cell-cell communication in the nervous system. The elaboration of a functional synapse requires both a specialized structure and an efficient communication system. For my thesis work, I studied proteins implicated in each of these functions: the structural molecules dystroglycan and dystrophin, and the signaling elements Insulin Receptor Substrate p58/53 and insulin receptor. The α/β-dystroglycan complex, believed to be the heart of cellmatrix adhesion in muscle and other tissues, provides a link between dystrophin, a cytoskeletal protein at the base of the muscle cell's Dystrophin Associated Protein Complex, and the extracellular matrix. In addition, dystrophin is found at central synapses, tightly associated with the postsynaptic density. The absence of dystrophin and the secondary loss of its associated proteins causes the genetic disease Duchenne Muscular Dystrophy. DMD affects both muscle and brain, causing a severe muscular dystrophy and lower IQs than control groups. In the first portion of my thesis work, I sought to determine the role of dystroglycan, dystrophin's peripheral partner, at central synapses. I probed Northern blots of brain regions to delineate the distribution of brain β-dystroglycan mRNA and to uncover any β-dystroglycan-related transcripts in brain. Then, using subcellular brain fractions, and cultured hippocampal neurons, I determined that whereas α-dystroglycan is associated with central synapses, β-dystroglycan is not. This discovery is surprising, and differs from the finding that dystrophin and α- and β-dystroglycan colocalize at the presynaptic membrane of retinal photoreceptors. In the course of the above mentioned work, using the anti-β-dystroglycan antiserum Ab98, I discovered a pair of proteins that were tightly associated with the postsynaptic density. These polypeptides of 58 kDa and 53 kDa (p58/53) were highly enriched in postsynaptic density (PSD) fractions from rat cerebral cortex, hippocampus, and cerebellum. In pursuit of a potential synapse-specific dystroglycan relative, I purified p58 and p53 by a combination of hydrophobic interaction chromatography and two-dimensional gel electrophoresis. Mass spectroscopy and peptide microsequencing revealed that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Whereas IRSp58/53 has no significant homology to β-dystroglycan other than the one span of peptides that confers its antibody cross-reactivity, its localization to the PSD newly implicates insulin signaling at synapses. Analysis of IRSp58/53 mass profiles, peptides, and mRNA indicated that IRSp58 and IRSp53 are the product of the same coding sequence. Immunolocalization showed that IRSp58/53 is expressed in the synapserich molecular layer of the cerebellum. Immunostaining of cultured hippocampal neurons showed that both IRSp58/53 and insulin receptor are highly concentrated at synapses. Like IRSp58/53, insulin receptors are a component of the PSD fraction. Together, these data suggest that the synapse is a specialized site for insulin signaling in the brain.

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

This thesis investigates the role of B cells as antigen-specific antigen-presenting cells (APC) in self tolerance to low concentrations of soluble self proteins and in acquired tolerance to low doses of soluble foreign protein antigens. Experiments were performed in normal and B cell-deficient animals, and tolerance induction was measured by T cell proliferation assays. T cell proliferation was reduced in B cell-deficient mice, indicating that B cells may be involved in efficient activation of naive T cells in response to protein antigen both in vivo and in vitro. To study acquired tolerance induced by low doses of soluble foreign protein antigen, normal and B cell-deficient adult mice were injected intravenously with repeated low doses (10 μg) of deaggregated ovalbumin (OVA), and then challenged with OVA in complete Freund's adjuvant. In animals treated with deaggregated OVA, the in vitro proliferative responses of LN T cells to OVA were significantly reduced, and production of the Th1 cytokine, IFN-γ, in response to OVA was lost. This occurred in both normal and B cell-deficient treated animals, indicating that B cell antigen presentation was not required for this phenomenon. B cells were also unnecessary for self tolerance of T cells to the transgenic self antigen, hen egg lysozyme (HEL), in a transgenic mouse strain with very low serum lysozyme concentration. Partial low zone tolerance induced by deaggregated, low-dose OVA was selective for the Th1 response, as measured by in vitro proliferation and IL-2 and IFN-γ production, because antibody responses of normal mice to this T cell-dependent antigen were largely unaffected. Both treated and untreated animals produced equivalent titers of anti-OVA antibodies, predominantly of the IgG1 and IgG2b isotypes, following challenge with OVA in complete Freund's adjuvant. Tolerance to low levels of the transgenic HEL self protein in mice expressing different MHC molecules was also addressed. Transgenic mice that were H-2b/b in the class II region were not tolerant to the transgenic self protein, whereas transgenic mice of the H-2b/k were tolerant.

The chicken hepatoma cell line, LMH, was evaluated with respect to its usefulness for studies of the regulation of heme metabolism. Levels of δ-aminolevulinate synthase mRNA arid accumulation of porphyrins were used to evaluate the heme biosynthetic pathway. Regulation of heme oxygenase-1 by known inducers was used as a measure of heme degradation. The induction of heme oxygenase-1 by sodium arsenite was characterized. AP-1 transcription factor elements and MAP kinase signal transduction pathways that modulate expression of endogenous heme oxygenase-1 and transfected heme oxygenase-1 reporter gene constructs in response to arsenite were delineated. In initial studies, the drug glutethimide was used alone or in combination with ferric nitrilotriacetate to induce δ-aminolevulinate synthase mRNA. Levels of porphyrins, intermediates in the heme biosynthetic pathway, and levels of δ-aminolevulinate synthase mRNA were increased by these treatments in a manner similar to those previously observed in the widely used model system, primary chick embryo liver cells. The iron chelator, deferoxamine, gave a characteristic shift in the glutethimide induced porphyrin accumulation in primary hepatocytes, but was found to have no, effect on LMH cells. Heme mediated repression of δ-aminolevulinate synthase mRNA levels was similar among primary hepatocytes and LMH cells. Heme oxygenase-1 was regulated by heme, metals, heat shock, and oxidative stress-inducing chemicals in LMH cells. Heat shock induction of heme oxygenase-1 mRNA levels was observed for the first time in primary chick embryo liver cells. These data supported the further use of LMH cells to elucidate mechanisms responsible for modulating heme oxygenase-1 gene expression in response to inducers. The remainder of the studies focused on the role of heme oxygenase-1 as a stress response protein. The oxidative stress inducer, sodium arsenite was used to probe the cellular mechanisms that control the expression of heme oxygenase-1. A series of promoter-reporter constructs were used to search the heme oxygenase-1 promoter for arsenite responsive elements. Several activator protein-1 (AP-1) transcription factor binding elements were identified by computer sequence analysis. Three of these sites, located at -1578, -3656, and -4597 base pairs upstream of the transcription start site, were mutated. The arsenite responsiveness of the reporter constructs containing mutated AP-1 elements was less than that of the same constructs containing wild type AP-1 elements. At least part of the arsenite-mediated induction of heme oxygenase-1 required the activity of AP-1 transcriptional elements. The MAP kinase signal transduction pathways and heme oxygenase-1 are activated by similar stimuli, including cellular stress. MAP kinases have been shown to exert control over gene expression through effects on the AP-1 family of transcription factors. The MAP kinases ERK, JNK, and p38 were activated by arsenite in LMH cells. Constitutively activated components of the ERK and p38 pathways increased expression of heme oxygenase-1 promoter-luciferase reporter constructs. Arsenite-mediated induction of heme oxygenase-1 was blocked by dominant negative ERK or p38 pathway components, and by specific inhibitors of MEK (upstream ERK kinase) or p38. In contrast, reporter gene expression was unchanged in the presence of constitutively activated JNK pathway components. Dominant negative JNK pathway components had no effect on arsenite induced heme oxygenase-1 gene activity. In summary, LMH cells were characterized as a new model system for the study of heme metabolism. This cell line was then used to delineate promoter elements and signaling pathways involved in the arsenite responsiveness of heme oxygenase-1 gene expression. Three AP-1 transcription factor binding sites in the heme oxygenase-1 promoter region were required for responsiveness to arsenite. The MAP kinases ERK and p38 were shown to play an integral role in arsenite-mediated induction of heme oxygenase-1. These studies elucidate one facet of heme oxygenase-1 regulation, and provide tools that will be useful in delineating additional regulatory mechanisms.

Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins. Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins. Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding. Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established. In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.

In humans, Bloom’s Syndrome is caused by a mutation of the RecQ helicase BLM. Patients with Bloom’s Syndrome exhibit a high amount of genomic instability which results in a high incidence of cancer. Though Bloom’s Syndrome has been intensively studied, there are still many questions about the function of BLM which need to be answered. While it is clear that loss of BLM increases genomic instability, the other effects of genomic instability on the organism aside from cancer such as a potential effect on aging, have yet to be elucidated. In Chapter II, I identify new phenotypes in the C. elegans ortholog of BLM, him-6. him-6 mutants have an increased rate of cell death, a mortal germ line phenotype, and an increased rate of mutations. Upon further examination of the mutator phenotype, it was determined that the increased rate of mutations was caused by small insertions and deletions. The mutator phenotype identified in him-6 mutants closely mimics the cellular phenotype seen in Bloom’s Syndrome cells. This indicates that HIM-6 may behave in a similar fashion to BLM. In addition to the mutator phenotype, it was found that loss of him-6causes a shortened life span. This may provide evidence that there is a link between genomic stability and aging. In Chapter III, I identify a new role for the transcription factor DAF-16. DAF-16 in C. elegans has been intensively studied and regulates a wide variety of pathways. In this chapter, I demonstrate via the well established unc-93 assay that loss of daf-16 causes a subtle mutator phenotype in C. elegans. This indicates that DAF-16 may play a role in suppression of spontaneous mutation. When I examined other classic genomic instability phenotypes, I found at 25°C, the number of progeny in the DAF-16 mutants was significantly reduced compared to wild type worms. Additionally, I demonstrate daf-16(mu86)has a cell death defect. This study identifies several new phenotypes caused by a loss of him-6. These phenotypes provide further evidence that loss of him-6 causes genomic instability. In addition, this study also demonstrates that him-6 has a shortened life span which may be due to genomic instability. Secondly, this study identifies a new role for DAF-16 in preventing the occurrence of spontaneous mutations. This may indicate a novel function for DAF-16 in maintaining genomic stability.