Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB.

Lysine acetylation is a post-translational modification (PTM) studied extensively in the context of histone proteins as a regulator of chromatin dynamics. Recent proteomic studies have revealed that as much as 10% of prokaryotic and mammalian proteins undergo lysine acetylation, and as such, the study of its biological consequences is rapidly expanding to include virtually all cellular processes. Unravelling the complex regulatory network governed by lysine acetylation will require an in depth knowledge of the lysine acetyltransferase enzymes that mediate catalysis, and moreover the development of methods that can identify enzyme-substrate relationships in vivo. This is complex task and will be aided significantly through the use of model organisms and systems biology approaches. The work presented in this thesis explores the function of the highly conserved NuA4 lysine acetyltransferase enzyme complex in the model organism Saccharomyces cerevisiae using systems biology approaches. By exploiting genetic screening tools available to the budding yeast model, I have systematically assessed the cellular roles of NuA4, thereby identifying novel cellular processes impacted by the function of the complex, such as vesicle-mediated transport and the stress response, and moreover identified specific pathways and proteins that are impacted by NuA4 KAT activity, including cytokinesis through the regulation of septin protein dynamics. Moreover, I have developed a mass spectrometry-based technique to identify NuA4-dependent acetylation sites amongst proteins that physically interact with NuA4 in vivo. Together this work demonstrates the diversity of processes impacted by NuA4 function in vivo and moreover highlights the utility of global screening techniques to characterize KAT function.

Obesity is an increasing health problem which is causally associated with insulin resistance and type 2 diabetes. Oxidative stress, i.e. overproduction of reactive oxygen species, is associated with insulin resistance and obesity and may be a major risk factor in the onset and progression of diabetes. Bernlohr Lab at University of Minnesota have study oxidative stress in adipocytes by silencing the enzyme glutathione S-transferase A-4 (GSTA4), an enzyme detoxifying 4-hydroxynonenal formed during oxidative stress. Their results indicate that lysine acetylation, an important post-translational modification, may be involved during oxidative stress. In this study lysine acetylation has been investigated in condition of oxidative stress in 3T3-L1 adipocytes and subcutaneous adipose tissue from mice using SDS-PAGE gel electrophoresis and western blot. Lysine acetylation was analyzed in different compartments of the cell such as in cytoplasm, mitochondria as well as in whole cell extracts. Silencing of GSTA4 and stimulation by TNF-α in 3T3-L1 adipocytes resulted in an increase of lysine acetylation in cytoplasm. Furthermore, stimulation by IL-6 did not have any effect on lysine acetylation. Surprisingly, subcutaneous adipose tissue from mice fed on a high-fat diet showed a decrease of lysine acetylation in cytoplasm compare to mice fed on a chow diet. In conclusion, lysine acetylation seems to change during oxidative stress and may be an important factor during insulin resistance, type 2 diabetes and obesity. Therefore, studying lysine acetylation and enzymes modulating acetylation may potentially increase our understanding of insulin resistance, type 2 diabetes and obesity and could lead to new therapies.

Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.

Tuberculosis continues to be one of the major causes of morbidity and mortality worldwide. Several mycobacterial species such as M. tuberculosis and M. africanum are responsible for causing this disease in humans. Reports of high cAMP levels in mycobacterial species (as compared to other bacteria such as E. coli) suggested that this second messenger may play an important role in the biology of mycobacteria. Further, it was reported that infection with mycobacteria led to an increase in the cAMP levels within the host macrophage. More recent studies have shown that this cAMP increase may be due to bacterially derived cAMP, hinting at a role for cAMP in mycobacterial pathogenesis. Given this background, the study of cAMP in mycobacteria proves to be an interesting field of research. Signalling through cAMP involves an interaction of this cyclic nucleotide with a cAMP-binding protein. These proteins typically contain a cyclic nucleotide-binding domain (CNB domain) linked to another (effector) domain. The CNB domain is thought to allosterically control the activity of the effector domain, thus mediating cellular responses to altered cAMP levels. For example, in the case of eukaryotic protein kinase A (PKA), binding of cAMP to the CNB domain results in relieving the inhibitory effects of the regulatory subunit on the catalytic subunit. The catalytic subunit then phosphorylates its target substrates, eliciting a variety of cellular responses. This work involves the characterisation of novel cAMP-binding proteins from mycobacteria, in an attempt to better understand cAMP signalling mechanisms in these organisms. The genome of M .tuberculosis H37Rv is predicted to code for ten CNB domain-containing proteins. One of these genes is Rv0998 (KATmt). KATmt was found to contain a GCN5 related N-acetyltransferase (GNAT) domain linked to a CNB domain. KATmt finds orthologues throughout the genus Mycobacterium, thereby suggesting its role in the basic physiology of these organisms. In addition, such a domain fusion is unique to mycobacteria and hence promises to deliver insights into the biology of this medically important genus. Presented here are the biochemical and functional characterisation of KATmt and its orthologue from M. smegmatis, MSMEG_5458 (KATms). Recombinant KATms bound cAMP with high affinity, validating the functionality of its CNB domain. Mutational and analogue-binding studies showed that the biochemical properties of the CNB domain were similar to mammalian protein kinase A and G-like CNB domains. The substrate for the GNAT acetyltransferase domain was identified to be a universal stress protein from M. smegmatis (MSMEG_4207). MSMEG_4207 was acetylated at a single lysine residue (Lys 104) by KATms in vitro. Further, cAMP binding to KATms increased the initial rate of acetylation of MSMEG_4207 by 2.5-fold, suggesting allosteric control of acetyltransferase activity by the CNB domain. To ascertain that KATms acetylated MEMEG_4207 in vivo, an in-frame deletion of the KATms gene was generated in M. smegmatis (ΔKATms). MSMEG_4207 was immunoprecipitated from wild-type M. smegmatis and the ΔKATms strains, followed by mass spectrometric analysis. Acetylated MSMEG_4207 was only present in the wild-type strain, confirming that KATms and MSMEG_4207 is an in vivo enzyme-substrate pair. Key biochemical differences were observed between KATms and KATmt. KATmt had an affinity for cAMP in the micromolar range, close to three log orders lower than that of KATms. In addition, KATmt showed strictly cAMP-dependent acetylation of MSMEG_4207. This demonstrates that orthologous proteins often evolve under varied selective pressures, resulting in divergent properties. Using a combination of bioluminescence resonance energy transfer (BRET) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), the conformational changes that occur upon cAMP binding to the CNB domain of KATms were monitored. A BRET-based conformation sensor was constructed for KATms by inserting KATms between GFP2 (green fluorescent protein) and Rluc (Renilla luciferase). An increase in BRET upon cAMP binding to the sensor was observed. HDXMS analysis revealed that besides the CNB domain, the only other region that showed conformational changes in KATms upon cAMP-binding was the linker region. To confirm that the linker region was important in propagating the effects of cAMP-binding to the acetyltransferase domain, an additional construct for BRET analysis encompassing the CNB domain and the linker region was generated. The magnitude of the increase in BRET was similar to the full length BRET-based sensor, validating the crucial role of the linker region in propagating cAMP-mediated conformational changes. A ‘PXXP’ motif found in the linker region, showed maximum exchange in HDXMS analysis. Mutation of both these proline residues to alanine in KATms, as well as KATmt, resulted in decoupling of cAMP-binding and allosteric potentiation of acetyltransferase activity. In contrast to the intricate parallel allosteric relays observed in other CNB domain-containing proteins, the CNB domain in KATms functions as a simpler cyclic nucleotide binding-induced switch involving stabilization of the CNB and linker domain alone. Therefore, KATms is an example of a primordial CNB domain where conformational changes are a consequence of binding-induced ordering alone. Using a computational approach, putative substrate proteins of KATmt from M. tuberculosis were identified. The substrate specificity of lysine acetyltransferases is determined loosely by a consensus sequence around the lysine residue which is acetylated. Using this property of protein acetyltransferases, the genome of M. tuberculosis H37Rv was mined for proteins harboring lysine residues in a similar sequence context as seen in MSMEG_4207. In vitro biochemical analysis of some of the predicted substrates helped confirm a subset of enzymes belonging to the fatty acyl CoA synthetase (FadD) class as substrates of KATmt. The acetylation of FadDs by KATmt was cAMP-dependent. In each of the four proteins tested, acetylation was found to occur at a single conserved lysine residue. To confirm that FadDs were acetylated by KATmt in vivo, BCG_1055, the orthologue of KATmt in M. bovis BCG, was deleted using the specialised transduction method. FadD13, one of the FadDs acetylated by KATmt in vitro, was immunoprecipitated from wild-type M. bovis and the ΔBCG_1055 strains using a FadD13-specific polyclonal antibody. Acetylated FadD13 was almost completely absent in ΔBCG_1055 but substantial amounts of acetylated FadD13 were present in the wild-type strain, indicating that FadD13 was indeed an in vivo substrate of KATmt. The functional consequences of acetylation of FadDs were analysed using an in vitro fatty acyl CoA synthetase assay. The activities of FadD2 and FadD13 were inhibited on acetylation with KATmt, while acetylation of FadD5 resulted in the formation of a novel product. Therefore, modification of the highly conserved lysine residue in these enzymes by acetylation led to loss or alteration of their enzymatic activity, suggesting that acetylation may be used as a regulatory mechanism to modulate the activities of some of the FadDs by KATmt in a cAMP-dependent manner. Given the extensive role of FadDs in cell wall biosynthesis and lipid degradation in mycobacteria, it seems possible that post-translational control by KATmt in a cAMP-dependent manner constitutes a novel mechanism utilised by these bacteria to regulate these pathways. This direct regulation of protein lysine acetylation by cAMP appears to be unique to mycobacteria, as orthologues of KATmt are not found outside this genus. In addition, the biochemical differences between KATmt and its orthologue from M. smegmatis KATms, indicate species specific variation, on a common theme. This study is the first report of protein lysine acetylation in mycobacteria. In addition to the identification of several proteins subject to this post-translational modification, the effect of acetylation on the enzymatic activities of some of them has been elucidated.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.