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Chromatin remodelers utilize the energy derived from ATP hydrolysis to mobilize nucleosomes. ISWI remodelers mobilize and evenly space nucleosomes to regulate gene expression. ISW2, an ISWI remodeler in yeast, has been shown to reposition nucleosome near promoter regions and represses both mRNA and antisense non coding RNA transcription. ISW2 is composed of four subunits and the catalytic Isw2 subunit consists of several conserved domains. The highly conserved ATPase domain is present at the N-terminus whereas the conserved HAND, SANT and SLIDE domain are towards the carboxyl terminal end of Isw2. Nucleosome mobilization by ISW2 requires both extranucleosomal DNA and the N-terminal tail of histone H4. DNA crosslinking and peptide mapping revealed that the ATPase domain contacts nucleosome two helical turns away (SHL2) from dyad to a site close to the H4 tail, whereas the HAND, SANT and SLIDE domain contact a 30bp stretch of DNA comprising the edge of nucleosome and ~20bp of extranucleosomal DNA. The ATPase domain and the C-terminal domains were investigated for their role in regulation of ISW2 activity both in-vitro and in-vivo. It appears that there are distinct modes of ISW2 regulation by these domains. Mutation of a patch of five acidic amino acids on the region of ATPase domain that contact SHL2 was found to be crucial for both ISW2 remodeling and nucleosome stimulated ATPase activity. Acidic patch mutant ISW2 was unable to mobilize nucleosome or hydrolyze ATP in absence of H4 tail. This indicates that the region of ATPase domain contacting nucleosome at SHL2 and H4 tail act in two separate and independent pathways to regulate ISW2 remodeling. Both HAND and SLIDE domain were shown to crosslink entry/exit site and linker DNA respectively. The roles of C-terminal domains were investigated either by deletion of the individual domain or mutation of conserved basic residues on the surface of these domains that are suspected to interact extranucleosomal with DNA. Deletion of HAND domain had minimal effect on in vitro ISW2 activity, however whole genome transcription analysis revealed one key role of this domain in ISW2 regulation. In absence of HAND domain, ISW2 had minimal role on repression of genes that were RPD3 (co-factor) dependent, however significantly derepressed genes that were RPD3 independent. At these loci, nucleosome positions were altered and ISW2 recruitment was reduced in absence of a functional HAND domain. Thus the HAND domain regulates recruitment and remodeling of ISW2 at those genes where ISW2 acts independent of other cofactors. The SANT domain, C-terminal to HAND domain, appears to control the "step size" of nucleosome remodeling and was found to be required for processive nucleosome remodeling by ISW2. Both H4 tail and SANT domain appear to control two distinct stages of ISW2 remodeling. A long alpha helical spacer separates SANT domain from SLIDE domain. SLIDE domain was found to be the protein-protein interaction domain that interacts with accessory Itc1 subunit to maintain ISW2 complex integrity. The two ways by which SLIDE domain regulate ISW2 is by binding or recruitment of ISW2 to promoter regions and additionally by binding independent regulation of both ATPase and remodeling activity. The remodeling mechanism of ISW2 was further compared with another ISWI type remodeler in yeast, Isw1a; using time resolved nucleosome remodeling combined with high resolution site specific histone DNA crosslinking at six different nucleosomal positions to track the movement of the nucleosomes. Nucleosome remodeled by the same remodeler showed discontinuous nucleosome movement between two tracking points indicating formation of small "bulges". One key difference in remodeling mechanism was that although both ISW2 and Isw1a moved nucleosomes towards longer linker DNA, only Isw1a remodeled nucleosomes "backtracked" ~11bp during remodeling. Backtracking of remodeling was prominently observed at nucleosomal regions in close proximity to translocase binding sites suggesting the potentially different mechanisms shared by similar remodeling complexes.

Indiana University-Purdue University Indianapolis (IUPUI) / The Ca2+ pump or Ca2+-ATPase of cardiac sarcoplasmic reticulum, SERCA2a, is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the apparent Ca2+ affinity of the enzyme. We propose that PLB decreases Ca2+ affinity by stabilizing the Ca2+-free, E2·ATP state of the enzyme, thus blocking the transition to E1, the high Ca2+ affinity state required for Ca2+ binding and ATP hydrolysis. The purpose of this dissertation research is to critically evaluate this idea using series of cross-linkable PLB mutants of increasing inhibitory strength (N30C-PLB < PLB3 < PLB4). Three hypotheses were tested; each specifically designed to address a fundamental point in the mechanism of PLB action. Hypothesis 1: SERCA2a with PLB bound is catalytically inactive. The catalytic activity of SERCA2a irreversibly cross-linked to PLB (PLB/SER) was assessed. Ca2+-ATPase activity, and formation of the phosphorylated intermediates were all completely inhibited. Thus, PLB/SER is entirely catalytically inactive. Hypothesis 2: PLB decreases the Ca2+ affinity of SERCA2a by competing with Ca2+ for binding to SERCA2a. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca2+-ATPase activity and phosphoenzyme formation were measured, and correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca2+ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca2+ for binding to SERCA2a. This was confirmed with the Ca2+ pump mutant, D351A, which is catalytically inactive but retains strong Ca2+ binding. Increasingly higher Ca2+ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB competes with Ca2+ for binding to the Ca2+ pump. Hypothesis 3: PLB binds exclusively to the Ca2+-free E2 state with bound nucleotide (E2·ATP). Thapsigargin, vanadate, and nucleotide effects on PLB cross-linking to SERCA2a were determined. All three PLB mutants bound preferentially to E2 state with bound nucleotide (E2·ATP), and not at all to the thapsigargin or vanadate bound states. We conclude that PLB inhibits SERCA2a activity by stabilizing a unique E2·ATP conformation that cannot bind Ca2+.

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The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo- iv PabABCE1 increases >30 fold from ~30 µM to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge () to being dependent on  with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1’s ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with v ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.

Rhodococcus equi is an important pathogen in foals between one to six months of age and is a major cause of death in in these animals. In addition, R. equi has recently emerged as a significant opportunistic pathogen in immunosuppressed humans, especially those infected with HIV. Despite the ability of the organism to survive stressful growth conditions, for example, exposure to elevated temperature and oxygen radicals, the role of heat shock proteins in the pathogenesis of R. equi has not been well documented. In this project we developed and optimised methods to purify the heat shock protein DnaK from R. equi, using a combination of ion-exchange and affinity chromatography. The effectiveness of the purification protocols were assessed using SDS-PAGE and Western-blotting with anti-DnaK antibodies, and the enzymic activity of the purified DnaK was verified with an ATPase assay. ATPase assays were also used to investigate the roles of other heat shock proteins in enhancing the activity of DnaK.

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