This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Thesis advisor: Charles Hoffman / The study of mammalian cAMP signaling has often been confounded by the fact that ten different genes encode adenylyl cyclases (ACs) that produce cAMP from ATP and 16 different genes encode phosphodiesterases (PDEs) that hydrolyze cAMP to AMP. In this study, mammalian AC cDNAs were cloned and integrated into strains of the fission yeast Schizosaccharomyces pombe that lack their endogenous AC to determine the basal activity of all ten AC isoforms. In addition, response to the stimulatory mammalian Gsα was determined by co-expression of a mutationally-activated form of the human GNAS1 gene. AC activity was assessed using an fbp1-GFP reporter that is repressed by cAMP production and PKA activity. Results confirm that all ten isoforms have detectable basal activity, and AC1-9 definitively respond to Gsα stimulation. When matched with a sufficiently potent mammalian phosphodiesterase (PDE), strains expressing mammalian ACs make good candidates for small molecule high throughput screening (HTS) to detect AC inhibitors. A 100,000 compound screen was recently performed to detect AC and Gsα inhibitors as well as PDE activators. A promising “hit” was progesterone, which has been previously suggested to inhibit ACs in Xenopus. Initial results suggest that progesterone inhibits AC1 and the closely-related AC3. These data demonstrate the utility of using S. pombe as an effective platform for identifying inhibitors of both basal and GNAS1-stimulated AC activity. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

<p>The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca²⁺, which has been shown to be due to influx of extracellular Ca²⁺ as a primary response.</p><p>Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX₁ than on OX₂ receptors.</p><p>When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca²⁺-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX₁ receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency G_S-coupling and a high-potency PKC coupling.</p><p>In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca²⁺-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.</p>

The orexin receptors are a pair of newly discovered G-protein coupled receptors which are activated by the neuropeptides orexins and play a role in sleep/vigilance, apetite/metabolism and neuroendocrine regulation. On a cellular level receptor activation results in, to name but a few effects, elevation of intracellular calcium and depolarisation. All cellular effects display an uncommon dependence of extracellular Ca2+, which has been shown to be due to influx of extracellular Ca2+ as a primary response. Here we provide evidence for a high specificity of orexin receptors for orexin peptides over other neuropeptides, despite previous reports of the opposite. Other neuropeptides could neither displace orexin-A from orexin receptors, nor affect functional responses induced by orexin peptides via orexin receptors. In an effort to assess the determinants of orexin-A binding to orexin receptors orexin-A was truncated/mutated and tested for functional responses. It was found that alterations in the orexin-A sequence had more prominent effects on the activation of OX1 than on OX2 receptors. When the signaling of orexin receptors was investigated in neuron-like cells it was found that they couple to Ca2+-metabolism and PLC activation in a manner similar to that in non-neuronal cells. Investigations of OX1 receptor regulation of adenylyl cyclases showed orexin receptors to have a dual effect on the production of cAMP. A high-affinity inhibitory coupling and a low-affinity stimulatory coupling. The stimulatory coupling was determined to consist of two components, a low potency GS-coupling and a high-potency PKC coupling. In conclusion we have shown that orexin receptors are preferentially activated by orexin peptides and the receptors couple to Ca2+-metabolism in a similar way in different contexts. Orexin receptors couple to both the phospholipase C and the adenylyl cyclase pathway and to some extent these pathways converge in the production of cAMP.

Structural investigations on two mycobacterial enzymes were carried out. Tuberculosis still remains a major threat to mankind even though drugs against it have been in use for many decades. The emergence of drug resistant strains of the bacteria calls for the identification of new targets based on which new drugs can be developed to combat the disease. A thorough understanding of the functioning of the target molecules is essential for this approach. We have taken up the structural studies on two such molecules, aspartic proteinases and adenylyl cyclases, of Mycobacterium tuberculosis with a view to obtain insights into their mechanisms of action at the atomic level. The work presented in the thesis includes (i) the identification, cloning, expression, purification and structure determination of a putative aspartic proteinase domain of M. tuberculosis and (ii) the crystal structure of an adenylyl cyclase of M. tuberculosis and its mutant; and also of an adenylyl cyclase from M. avium. Chapter 1 presents an overview of aspartic proteinases and nucleotide cyclases with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Work on the putative aspartic proteinase domain identified in M. tuberculosis is presented in Chapter 3. The structure of the aspartic proteinase domain is the first structural report of such domain from any bacteria. A search in the genome of M. tuberculosis showed a weak similarity to the HIV aspartic proteinase sequence. This region corresponds to the C-terminal domain of a PE family protein in M. tuberculosis. The presence of two signature motifs, DTG and DSG, of aspartic proteinases in the full sequence of this domain encouraged us to take up further studies on this domain. Previous reports identifying the protein as a surface antigen and our findings on the occurrence of similar domains in two other PE proteins of M. tuberculosis and also in other pathological strains of Mycobacteria indicated that these domains probably play an important role in infecting the host. The crystal structure of one of the domains showed that it has a pepsin-like fold and the catalytic site architecture of known aspartic proteinases. However, no proteolytic activity was detected. The size of the molecule is intermediate to eukaryotic pepsins and the homodimeric retroviral pepsins. A close examination of the binding site revealed subtle differences when compared to the active enzyme structures. Appropriate mutations of some of the residues in this region to convert it to an active enzyme did not make it active. Once the in vivo function of these putative aspartic proteinase domains is established, their potential to act as drug targets can be probed as the PE proteins are present exclusively in pathogenic Mycobacteria. As part of an ongoing project on adenylyl cyclases of Mycobacteria, we have taken up the structure analysis of the catalytic domains of two adenylyl cyclases; Rv1625c from M. tuberculosis and Ma1120 from M. avium. This work is described in Chapter 4. The wild-type of Rv1625c crystallized as a domain swapped head to head inactive dimer even though it is an active dimer in solution and expected to have a head to tail arrangement as in the previously reported structures of the active forms of the enzyme. Mutation of a phenylalanine residue presumed to occur at the subunit interface of the active dimeric structure of the enzyme to an arginine residue, a conserved residue of guanylyl cyclases, resulted in reduced adenylyl cyclase activity. This mutant crystallized as a monomer though it was expected to be an active dimer. Similarly, Ma1120 also has a monomeric structure in the crystal in spite of showing activity in solution. Though our aim was to capture the active dimers in the crystalline state we did not succeed in this effort in any of the three cases. The catalytic reaction probably takes place very rapidly with the formation of a transient active form of the dimer which cannot be easily crystallized. However, the analysis revealed new structures which are likely to represent the stable states of the enzyme when it is required to stay inactive in certain conditions. We have also established that the N-terminal segments of the enzyme, a loop at the dimeric interface and external factors like pH are involved in determining the oligomeric status of the enzyme thereby regulating its function. Publications 1 Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16; Deivanayaga V. Barathy and K. Suguna; FEBS Open Bio (In Press) 2 New structural forms of mycobacterial adenylyl cyclases (in preparation)

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-157).

Mycobacterium tuberculosis is one of the most successful human pathogens, estimated to have infected close to one-third of the global human population. In order to survive within its host, M. tuberculosis utilises multiple signalling strategies, one of them being synthesis and secretion of universal second messenger cAMP. This process is enabled by the presence of sixteen predicted adenylyl cyclases in the genome of M. tuberculosis H37Rv, ten of which have been characterised in vitro. The synthesized cAMP is recognised by ten putative cAMP-binding proteins in which the cyclic AMP-binding domain is associated with a variety of enzymatic domains. The cAMP signal can be extinguished by degradation by phosphodiesterase’s, secretion into the extracellular milieu or via sequestration of the nucleotide by upregulation of a high-affinity cAMP-binding protein. Of the sixteen adenylyl cyclases (ACs) encoded by M. tuberculosis H37Rv, a subset of multidomain adenylyl cyclases remain poorly characterised, primarily due to challenges associated with studying these in vitro. The adenylyl cyclase domain in these proteins is associated with an NB-ARC domain (nucleotide binding domain common to APAF-1, plant R proteins and CED-4), a TPR domain (tetratricopeptide repeat) and an LuxR-type HTH motif (helix-turn-helix). This architecture places these multidomain mycobacterial ACs within a larger group of STAND (Signal transduction ATPase’s with numerous domains) proteins, and hence they will be referred to as STAND ACs. The STAND proteins are a recently recognised class of multidomain ATPases which integrate a variety of signals prior to activation. Activation is accompanied by formation of large oligomeric signalling hubs which facilitate downstream signalling events. While most STAND proteins have a single effector domain followed by an NB-ARC domain and a scaffolding domain, the STAND ACs distinguish themselves by retaining two effector domains, the AC domain and the HTH domain, at the N- and C- termini respectively. The cyclase, NB-ARC, TPR and HTH domains have widely divergent taxonomic distributions making the presence of these four domains in a single polypeptide rare. In fact, proteins with cyclase-NB-ARC-TPR-HTH (C-A-T-H) domain organisation were found to be encoded almost exclusively by slow growing mycobacterial species, a clade that harbours most mycobacterial pathogens, such as M. tuberculosis and M. leprae. Notably, one of the STAND ACs, Rv0386, is the only mycobacterial AC shown till date to be required for virulence of M. tuberculosis in mice. Using phylogenetic, the evolutionary underpinnings of this domain architecture were examined. The STAND ACs appear to have most likely evolved via a domain gain event from a cyclase-ATPase-TPR progenitor encoded by a strain ancestral to M. marina. Subsequently, the genes duplicated and diverged, sometimes leading to frameshift mutations splitting the cyclase domain from the C-terminal domains. Consequently, M. tuberculosis encodes for three ‘full-length’ STAND ACs, namely, Rv0386, Rv1358 and Rv2488c and one split STAND AC. The split STAND AC is made up of Rv0891c, containing the AC domain, and Rv0890c, containing the NB-ARC, TPR and HTH domains. rv0891c and rv0890c were found to be expressed as an operatic transcript, though they were translationally uncoupled. Pertinently, M. Canetti, an early-branching species of the M. tuberculosis complex, contains an orthologue of Rv0891c and Rv0890c where all four domains are present in a single polypeptide. Sequence analysis of the four STAND ACs in M. tuberculosis allowed predictions of significant divergence in function. These proteins showed high sequence conservation in their HTH domains, with substantial sequence divergence in their TPR, NB-ARC and AC domains. Biochemical analysis on the AC domains revealed that Rv0891c and Rv2488c possessed poor or no AC activity, respectively. On the other hand, the cyclase domain of Rv0386 could catalyse cAMP synthesis. Moreover, for both Rv0891c and Rv0386, presence of the C-terminal domains potentiated adenylyl cyclase activity, suggestive of allosteric regulation within the STAND AC module. Studies on Rv0891c also revealed that the protein could inhibit the adenylyl cyclase activity of Rv0386 in trans. This result thus provided a novel mechanism by which proteins harbouring poorly active/inactive adenylyl cyclase domains could contribute to cAMP levels, by acting as inhibitors of other adenylyl cyclases. The STAND ACs were found to be inactive ATPases. Additionally, incubation with nucleotides did not stimulate oligomerisation of these proteins, unlike what has been shown for several other STAND proteins. However, mutations in the NB-ARC domain perturbed the basal oligomeric state of these proteins, indicating that the NB-ARC domain can influence self- association. A subset of NB-ARC domain mutants also showed increased adenylyl cyclase activity, reiterating the inter-domain cross-talk in the STAND ACs. Since the AC activity of these proteins was meagre, the properties of the HTH domain were examined, as an alternative effector domain. Genomic SELEX was performed using the TPR-HTH domains of Rv0890c, and revealed a set of sequences that bound to this protein, though they lacked common sequence features. Further analysis revealed that Rv0890c bound to DNA in a sequence-independent manner, through the HTH domain. This binding was cooperative with multiple protein units engaging in DNA-binding. Due to the cooperative nature of binding and the lack of sequence preference, Rv0890c appeared coat the DNA molecule. This was further proved by the ability of Rv0890c to protect DNA from DNaseI-mediated degradation, and the requirement for long DNA sequences to form stable DNA-protein complexes. Studies also revealed that Rv0890c interacted with RNA and ssDNA. In fact, the protein as purified from heterologously expressing E. coli cells was bound to RNA. RNA-binding by a LuxR-type HTH has not been reported previously, providing a new function for this class of HTHs. Interestingly, nucleic acid-binding by a fusion Rv0891c-Rv0890c protein, similar to the one encoded in M. canetti, was shown to stimulate adenylyl cyclase activity. This was likely due to a relief of inhibitory interactions between the TPR-HTH and the AC domains, on DNA-binding. Given the high sequence similarity between the HTH domains of the STAND ACs, they were expected to bind to DNA in an identical manner. Indeed, the HTH domains of Rv0386 and Rv1358 engaged with DNA with an identical affinity as Rv0890c. Sequence comparisons in the HTH domain enabled identification of conserved basic residues, of which one, R850 was essential for nucleic acid-binding. Surprisingly however, Rv0386 and Rv1358 did not exhibit RNA-binding, pointing towards functional divergence of Rv0890c from its paralogues. Since the HTH domains of the STAND ACs were highly conserved, it was possible that the ability to bind to RNA was instead dictated by the adjacent TPR modules. To examine this possibility, TPR domains were swapped between Rv0890c and Rv0386. Interestingly, both the chimeric proteins showed a reduced ability to bind to DNA, while showing a complete absence of RNA- binding. These results suggested that the TPR domains were critical in modulating nucleic acid-binding. Moreover, the effect of the TPR domain was context-dependent, since the presence of non-cognate TPR domains hampered nucleic acid-binding. However, the ability to bind to RNA was not solely governed by the TPR domain since the Rv0890cTPR-Rv0386HTH chimeric protein did not show RNA-binding, in spite of containing a permissive TPR domain. To further dissect the molecular requirements for RNA-binding, the conservation of basic residues between the HTH domains of Rv0890c versus Rv1358 and Rv0386 was examined. Interestingly the HTH domain Rv0890c contained two additional positively charged residues over Rv1358 and Rv0386. Mutations of these abolished RNA-binding by Rv0890c. Thus the evolution of two basic residues permit Rv0890c to diverge in its nucleic acid-binding properties, a possible example of defunctionalisation following gene duplication. In summary, this thesis attempts to understand the evolution and functions of the STAND ACs, a group of pathogenically relevant and uniquely mycobacterial multidomain proteins. Phylogenetic analysis revealed an expansion of this gene family in slow growing mycobacteria. Biochemical characterisation showed that following gene duplication, the resulting proteins diverge both in their ability to synthesize cAMP and in their association with nucleic acids. Studies on these proteins also revealed novel mechanisms of regulation of mycobacterial cAMP levels. Additionally, these proteins exhibited indiscriminate binding to DNA/nucleic acids indicating that they may be responsible for global functions in the cell which extend beyond cAMP synthesis.