The overall emphasis in this thesis is the study of the regulation of virus infections by natural killer (NK) cells. In initial analyses, vaccinia virus (VV)-infected cells were found to be more sensitive to NK cell-mediated lysis during a discrete period of time post-infection. This enhanced susceptibility to lysis correlated with enhanced triggering (but not binding) of the effector cells and a concomitant decrease in target cell H-2 class I antigen expression. Furthermore, VV-infected cells became resistant to lysis by allospecific cytotoxic T lymphocytes (CTL) at a time when they were very sensitive to killing by NK cells or VV-specific CTL. This suggested that alterations in class I MHC antigens may affect target cell sensitivity to lysis by NK cells. The hypothesis that viral peptide charging of H-2 class I molecules can modulate target cell sensitivity to NK cell-mediated lysis was tested by treating target cells with synthetic viral peptides corresponding to the natural or minimal immunodominant epitopes defined for virus-specific CTL, and then target cell susceptibility to NK cell-mediated lysis was assessed. None of the 12 synthetic viral peptides used were able to significantly alter target cell lysis by NK cells under any of the conditions tested. In order to determine if H-2 class I molecules were required in the regulation of a virus infection by NK cells in vivo, intact or NK depleted (treated with anti-asialo GM1 antiserum) β2-microglobulin-deficient [β2m (-/-)] mice, which possess a defect in H-2 class I antigen expression, were infected with the prototypic NK-sensitive virus, murine cytomegalovirus (MCMV). In anti-asialo GM1-treated β2m (-/-) mice, as well as in β2m + (H-2 class I normal) control mice also treated with anti-asialo GM1 a significant enhancement in splenic MCMV titers as compared to NK-intact animals, was observed. When thymocyte expression of H-2 class I molecules (H-2Db) in normal mice was analyzed, it was found that following MCMV infection, H-2Db expression was significantly greater than the low level of expression found in uninfected thymocytes. In marked contrast, thymocytes from β2m (-/-) mice did not display any detectable H-2Db before or after infection. These in vivoresults demonstrate that NK cells can regulate a virus infection, at least in the case of MCMV, independent of H-2 class I molecule expression. Thymocytes from uninfected normal mice were found to be very sensitive to NK cell-mediated lysis, whereas those from MCMV-infected animals were completely resistant, presumably due to the protective effects of MCMV-induced interferon (IFN). However, thymocytes from MCMV-infected β2m (-/-) mice were only slightly protected from lysis by NK cells, consistent with the inverse correlation between MHC class I antigen expression and sensitivity to NK cell-mediated lysis. These results provide in vivoevidence suggesting a requirement for MHC class I molecules in IFN-mediated protection from lysis by NK cells. In addition to the analysis of H-2 class I molecules on target cells, the identity of a molecule present on the surface of all NK cells and other cytotoxic effector cells, which is recognized by a monoclonal antibody (mAb) generated in this laboratory designated CZ-1, and can also modulate NK cell triggering, was also of interest. This laboratory has previously reported that this antigen is upregulated on cytotoxic (and other) lymphocytes following a virus infection in vivo, or upon activation in vitro. Using competitive FACS analysis and fibroblasts transfected with various isoforms of CD45, it was found that mAb CZ-1 recognizes a sialic acid-dependent epitope associated with a subpopulation of CD45RB molecules.

P22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.

The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation? The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro. This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.

The yeast SWI/SNF complex is the prototype of a subfamily of ATP-dependent chromatin remodeling complexes. It consists of eleven stoichiometric subunits including Swi2p/Snf2p, Swi1p, Snf5p, Swi3p, Swp82p, Swp73p, Arp7p, Arp9p, Snf6p, Snf11p, and Swp29p, with a molecular weight of 1.14 mega Daltons. Swi2p/Snf2p, the catalytic subunit of SWI/SNF, is evolutionally conserved from yeast to human cells. Genetic evidence suggests that SWI/SNF is required for the transcriptional regulation of a subset of genes, especially inducible genes. SWI/SNF can be recruited to target promotors by gene specific activators, and in some cases, SWI/SNF facilitates activator binding. Biochemical studies have demonstrated that purified SWI/SNF complex can hydrolyze ATP, and it can use the energy from ATP hydrolysis to generate superhelical torsion, mobilize mononucleosomes, enhance the accessibility of endonucleases to nucleosomal DNA, displace H2A/H2B dimers, induce dinucleosome and altosome formation, or evict nucleosomes. A human homolog of Swi2p/Snf2p, BRG1, is the catalytic subunit of the human SWI/SNF complex. Interestingly, isolated BRG1 alone is able to remodel a mononucleosome substrate. Importantly, mutations in mammalian SWI/SNF core subunits are implicated in tumorigenesis. Therefore, it remains interesting to characterize the role(s) of each subunit for SWI/SNF function. In this thesis project, I dissected SWI/SNF chromatin remodeling function by investigating the role of the SANT domain of the Swi3p subunit. Swi3p is one of the core components of SWI/SNF complex, and it contains an uncharacterized SANT domain that has been found in many chromatin regulatory proteins. Earlier studies suggested that the SANT domain of Ada2p may serve as the histone tail recognition module. For Swi3p, a small deletion of eleven amino acids from the SANT domain caused a growth phenotype similar to that of other swi/snf mutants. In chapter I, I have reviewed recent findings in the function of chromatin remodeling complexes and discuss the molecular mechanism of their action. In chapter II, I characterized the role of the SANT domain of Swi3p. I found that deletion of the SANT domain caused a defect in a genome-wide transcriptional profile, SWI/SNF recruitment, and more interestingly impairment of the SANT domain caused the dissociation of SWI/SNF into several subcomplexes: 1) Swi2p/Arp7p/Arp9p, 2) Swi3p/Swp73p/Snf6p, 3) Snf5p, and 4) Swi1p. Artificial tethering of SWI/SNF onto a LacZ reporter promoter failed to activate the reporter gene in the absence of the SANT domain, although Swi2p can be recruited to the LacZ promoter. We thus demonstrated that the Swi3p SANT domain is critical for Swi3p function and serves as a protein scaffold to integrate these subcomplexes into an intact SWI/SNF complex. In Chapter III, I first characterized the enzymatic activity of the subcomplexes, especially the minimal complex of Swi2p/Arp7p/Arp9p. We found that this minimal subcomplex is fully functional for chromatin remodeling in assays including cruciform formation, restriction enzyme accessibility in mononucleosomal and nucleosomal array substrates, and mononucleosome mobility shift. However, it is defective in ATP-dependent removal of H2A/H2B dimers. Moreover, we found that Swi3p and the N-terminal acidic domain of Swi3p strongly interact with GST-H2A and H2B but not GST-H3 or H4 tails. We purified a SWI/SNF mutant (SWI/SNF-Δ2N) that lacks 200 amino acids within the N-terminal acidic domain of Swi3p. Intriguingly, SWI/SNF-Δ2N failed to catalyze ATP-dependent dimer loss, although this mutant SWI/SNF contains all the subunits and has intact ATP-dependent activity in enhancing restriction enzyme accessibility. These data help to further understand the molecular mechanism of SWI/SNF, and show that H2A/H2B dimer loss is not an obligatory consequence of ATP-dependent DNA translocation, but requires the histone chaperone function of the Swi3p subunit. Based on these findings, we proposed a new model of the structural and functional organization of the SWI/SNF chromatin remodeling machinery: SWI/SNF contains at least four distinct modules that function at distinct stages of the chromatin remodeling process. 1) Swi1p and Snf5p modules directly interact with gene specific activators and function as the recruiter; 2) Swi2p/Arp7p/Arp9p generates energy from ATP hydrolysis and disrupts histone/DNA interactions; and 3) Swi3p/Swp73p/Snf6p may play dual roles by integrating each module into a large remodeling complex, as well as functioning as a histone H2A/H2B chaperone to remove dimers from remodeled nucleosomes. Chapter IV is a perspective from current work in this project. I first discuss the interest in further characterizing the essential role of Snf6p, based on its activation of LacZ reporter on its own. Using in vitro translated protein and co-IP studies, I tried to pinpoint the requirement of the SANT domain for SWI/SNF assembly. I found that Swi3p directly interacts with Swp73p, but not with other subunits. When Swi3p is first incubated with Swp73p, Swi3p also interacts with Snf6p, indicating that Swi3p indirectly interacts with Snf6p, therefore forming a subcomplex of Swi3p/Swp73p/Snf6p. This subcomplex can also be reconstituted using in vitro co-translation. Consistent with the TAP preparation of this subcomplex, partial deletion of the SANT domain of Swi3p does not affect the assembly of Swi3p/Swp73p/Snf6p in vitro. However, the assembly of SWI/SNF complex was not detected in the presence of eight essential in vitro translated subunits or from co-translation of all the subunits. I have discussed the interest in further characterizing the histone chaperone role of the Swi3p N-terminal acidic domain and the role of other core subunits of SWI/SNF such as Snf6p for transcriptional regulation.

The regulation of protein function through oligomerization is a common theme in biological systems. In this work, I have focused on the effects of the oligomeric states of the human Rad52 protein on activities related to DNA binding. HsRad52, a member of the RAD52 epistasis group, is thought to play an important and as yet undefined role in homologous recombination. HsRad52 preferentially binds to ssDNA over dsDNA and stimulates HsRad51-mediated strand exchange (Benson et al., 1998). In either the presence or absence of DNA, HsRad52 has been observed to form both 10 nm ring-like structures as well as higher order oligomers consisting of multiple 10 nm rings (Van Dyck et al., 1998; Van Dyck et al., 1999). Earlier protein-protein interaction studies mapped the domain responsible for HsRad52 self-association in the N-terminus (residues 85-159) (Shen et al., 1996). Data presented here identifies a novel self-association domain in the C-terminus of HsRad52 that is responsible for the formation of higher order oligomers. VanDyck et al. observed DNA ending binding complexes consisting of multiple rings (Van Dyck et al., 1999). They proposed that these higher order oligomers may be functionally relevant. In this work, we demonstrate that DNA binding depends on neither ring shaped oligomers nor higher order oligomers but that activities of HsRad52 that require simultaneous interaction with more than one DNA molecule depend on the formation of higher order oligomers consisting of multiple HsRad52 rings. Early studies of HsRad52 proposed that the DNA binding domain resides in the highly conserved N-terminus of the protein (Park et al., 1996). A series of studies using truncation mutants of HsRad52 have provided evidence that supports this hypothesis. For example, we demonstrated that a truncation mutant containing only the first 85 residues of the protein is still able to bind DNA (Lloyd, submitted 2002). In this study, we demonstrate that aromatic (Y65, F79 and Y81) and hydrophobic (L43, I52 and I66) residues within the N-terminus contribute to DNA binding by either directly contacting the DNA or by stabilizing the structure of the protein. In summary, through the work presented in this dissertation, we have determined that the formation of 10 nm rings is mediated by a self-association domain in the N-terminus and that the formation of higher order oligomers consisting of multiple HsRad52 rings is mediated by an additional self-association domain in the C-terminus. We have correlated the oligomeric properties of HsRad52 with its biochemical functions related to DNA binding. Additionally, we have demonstrated that aromatic and hydrophobic residues contribute to DNA binding. Further studies will differentiate between the contribution of these residues to the DNA binding by stabilizing the overall structure of the protein versus making specific DNA contacts. Additional studies will also address how the oligomeric state of HsRad52 contributes to its role in HsRad51-mediated strand exchange.

The photon receptor, rhodopsin, and the GTP-binding regulatory protein, transducin, belong to a family of G protein-coupled receptors. The activation process through which guanine nucleotide exchange of the G protein is accomplished was investigated utilizing these components of the visual transduction system. Rhodopsin, modelled as an enzyme in its interaction with substrates, transducin and guanine nucleotides, was characterized to catalyze the G protein's activation by a double-displacement mechanism. Remarkable allosteric behavior was observed in these kinetic studies. Equilibrium binding studies were performed to investigate the molecular basis of the positive cooperative behavior between transducin and rhodopsin. These experiments show that the origins of the allosterism must arise from oligomeric assemblies between receptor and G protein. The determined Hill coefficient, nH = 2, suggests that at least two transducin molecules are involved, and the Bmax parameter a1so indicates that multimeric assemblies of rhodopsin may participate in the positive cooperative interactiions. Physical studies of transducin in solution were performed and do not indicate the existence of a dimeric structure, in contrast to the kinetic and binding experiments which analyze interactions at the membrane surface. Since the latter environment represents the native surroundings in vivo, aspects of the allosteric behavior must be considered for a complete understanding of the signal transduction mechanism. The reported findings are interpreted in the context of homologies between other G protein-coupled receptor systems in order to develop a model for the molecular basis of the mechanism and regulation of this mode of signal transduction.

Activation mechanism of the α-factor pheromone receptor of Saccharomyces cerevisiae was analyzed using biochemical and genetic techniques. An in vitro partial proteolysis assay was developed to determine the conformational change of the receptor that occurs upon binding of agonist. The activation specific cleavages were established by comparing cleavage products with antagonist versus agonist occupied receptor. Of the changes in peptide pattern that were revealed by trypsinization, the fragment resulting from the exposure of the third loop to the protease was found to be agonist specific and to be G-protein independent. A low-affinity binding receptor mutant was isolated which failed to undergo this agonist induced conformational change. Four intra-allelic suppressors of this receptor mutant were isolated and all were mapped to the ends of transmembrane helices 4, 5, 6 and 7; all were found to be replacements of non-polar residues by polar ones. The role of the suppressor mutations in conformational change was analyzed.

Cells as dynamic, interactive, and self contained units of life have a need for molecular motors that can create physical forces to move cargoes within the cell. Cytoplasmic dynein 1 is one such molecular motor that has many functions in the cell. The number and variety of functions that involve cytoplasmic dynein 1 suggest that there are a number of different binding sites on dynein for different proteins. Cytoplasmic dynein 1 is a multiprotein complex made up of six different subunit families. The many different combinations of subunits that could be used to make up a cytoplasmic dynein 1 holocomplex provides the variety of different binding sites for cargoes that can be individually regulated. The following chapters flush out how light intermediate chain 1 (LIC1), a subunit of cytoplasmic dynein 1, is involved with multiple dynein functions involving the binding of different cargoes to the cytoplasmic dynein 1 holocomplex, and how the binding of these cargoes can be regulated. First, LIC1 is found to be involved in the spindle assembly checkpoint. LIC1 appears to facilitate the removal of Mad1-Mad2, a complex important in producing a wait anaphase signal, from kinetochores. Second, the involvement of LIC1 in the spindle assembly checkpoint requires the phosphorylation of LIC1 at a putative Cdk1 phosphorylation site. This site is located in a domain of LIC1 that binds various proteins suggesting that this phosphorylation could also regulate these interactions. Third, LIC1 is involved in the centrosomal assembly of pericentrin, an important centrosomal protein. From the data presented herein, LIC1 is shaping up as a multifunctional cargo binder for cytoplasmic dynein 1 that requires regulation of its various cargoes.

Synthesis of histone proteins occurs largely during the S phase of the cell cycle and coincides with DNA replication to provide adequate amounts of histones necessary to properly package newly replicated DNA. Controlling transcription from cell cycle dependent and proliferation specific genes, including histone H4, is an important level of regulation in the overall governance of the cell growth process. Coordination of histone gene transcription results from the cumulative effects of cell signaling pathways, dynamic chromatin structure and multiple transcription factor interactions. The research of this dissertation focused on the characterization and identification of transcription factors interacting on the human histone H4 gene FO108. I also focused on the elucidation of regulatory elements within the histone coding region. Our results suggest a possible mechanism by which a transcription factor facilitates reorganization of histone gene chromatin structure. The histone promoter region between -418 nt and -215 nt, Site III, was previously identified as both a positive and negative cis-regulatory element for transcription. Results of in vitroanalyses presented in this dissertation identified multiple transcription factors interacting at Site III. These factors include H4UA-1/YY1, AP-2, AP-2 like factor and distal factor (NF-1 like factor). Transient transfection experiments show that Site III does not confer significant influence on transcription; however, there may exist a physiological role for Site III which would not be detected in these assay systems. We analyzed the histone H4 gene sequences for additional transcription factor binding motifs and identified several putative YY1 binding sites. Using electrophoretic mobility shift assays (EMSA), we found that Site IV, Site I and two elements within the histone H4 coding region are capable of interacting with YY1. In transient transfection experiments using reporter constructs containing either Site III or one of the coding region elements as potential promoter regulatory elements, and an expression vector encoding YY1, we observed levels of expression up to 2.7 fold higher than from the reporters lacking these elements. Therefore, YY1 appears to interact at multiple regulatory sites of the histone gene and can influence transcription through these elements. Prior to this study, the role of the coding region in histone gene expression was not known. To determine if the coding region is involved in regulating transcription, I constructed and tested a series of heterologous reporter constructs containing various sequences of the histone coding region. Results from these experiments demonstrated that the histone coding region contains three repressor elements. Extensive in vitro analysis indicated that the three repressor elements interact with the repressor CDP/cut. Further analysis showed that CDP/cut interactions with the repressor elements are cell cycle regulated and proliferation specific. CDP/cut interactions increase during the cell cycle when histone transcription decreases. These observations are consistent with the hypothesis that CDP/cutis a cell cycle regulated repressor factor which influences transcription of the histone H4 gene as such. The proximal promoter region of the histone H4 gene between -70 nt and +190 nt is devoid of normal nucleosome structure. This same region contains multiple CDP/cut binding sites. We hypothesized that CDP/cut is involved with chromatin remodeling of the histone gene. DNase I footprinting and EMSA results show purified recombinant CDP/cut interacts specifically with the histone promoter reconstituted into nucleosome cores. Thus, CDP/cutmay facilitate the organization of chromatin of the histone gene. In conclusion, the research presented in this dissertation supports the hypothesis that expression from the human histone H4 gene FO108 is regulated by multiple cis-regulatory elements which interact with several proteins. CDP/cut interacts with Site II, the three repressor elements in the histone coding region and at Distal Site I. YY1 interacts at Site IV, Site III, Site I, and twice in the coding region. ATF/CREB interacts with Site IV and Site I. Distal factor interacts with Site III and within the histone coding region. IRF 2 interacts with Site II and Distal Site I. Thus, histone gene expression is probably regulated by transcription factors CDP/cut, YY1, IRF 2 and ATF/CREB interacting with multiple regulatory elements dispersed throughout its promoter and the coding region. Cell cycle regulation of these transcription factors may contribute to cell cycle dependent expression of the histone gene.

The nervous system requires rapid, efficient, and accurate transmission between cells for proper functioning. Synapses are the predominant structures through which such vital communication occurs. How synapses are formed, maintained, and eliminated are questions of fundamental importance. At the nerve-muscle synapse, formation of the postsynaptic apparatus is directed by agrin. The hallmark activity of agrin is the aggregation of acetylcholine receptors (AChRs) into dense clusters opposite the presynaptic nerve terminal. Early events in the agrin signal transduction cascade include activation of the receptor tyrosine kinase MuSK and tyrosine phosphorylation of AChRs, but how these events lead to AChR cluster formation is unknown. Using the calcium buffer BAPTA, we demonstrate that intracellular calcium fluxes are necessary for agrin-induced formation of AChR clusters. However, clamping calcium fluxes before agrin stimulation does not alter agrin-induced phosphorylation of either MuSK or AChRs, indicating that this calcium-dependent step occurs downstream of both MuSK and AChR phosphorylation. These results identify a new step in the agrin signaling pathway required for the formation of AChR clusters. We show that intracellular calcium fluxes also play an important role in stabilizing AChR clusters. Clamping intracellular calcium fluxes results in rapid dispersal of AChR clusters and dephosphorylation of both MuSK and AChRs, even if agrin is continually present. Furthermore, the protein tyrosine phosphatase inhibitor pervanadate inhibits both the dispersal and dephosphorylation, indicating a role for a tyrosine phosphatase in AChR cluster dispersal. Our data indicate that AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes that tonically inhibit a tyrosine phosphatase localized to AChR clusters. Our findings also show that distinct molecular mechanisms mediate the formation and the dispersal of agrin-induced AChR clusters. The work presented here expands our understanding of synaptic differentiation in several ways. First, I characterized a new, calcium-dependent step required for the formation of agrin-induced AChR clusters. Next, I showed that postsynaptic specializations must be actively maintained, and describe a molecular mechanism that stabilizes AChR clusters. Finally, dispersal and formation of AChR clusters occurs by distinct pathways. Our understanding of the mechanisms regulating the formation and modulation of synapses will help us to better understand how the nervous system develops and responds to the world around us.