The signaling pathways of tyrosine kinase Trk receptors and their downstream biological effects are well known, but the parameters controlling the interactions between the receptors and their natural ligands still remain to be defined. Recent published reports from our laboratory indicate that nerve growth factor (NGF)-induced TrkA receptor activation is dependent on a membrane cellular sialidase. This sialidase activity specifically targets and hydrolyzes sialyl α-2, 3-linked β-galactosyl residues resulting in the desialylation and activation of the receptor. These findings support a novel hypothesis that places mammalian sialidase(s) in a cycle of activation of these receptors by their natural ligand. Taken together, they also predict a prerequisite desialylation of Trk receptors caused by a sialidase on the cell surface enabling the removal of a steric hindrance to receptor dimerization. Until now, the sialidase associated with neurotrophin-treated live Trk-expressing cells has not been identified. The molecular mechanism(s) of sialidase activation by neurotrophin factors binding to their receptors also remains unknown. In this thesis, the novel role of Neu1 sialidase in the activation of ligand-induced TrkA and TrkB receptors has been identified. It has been reported for the first time that Neu1 is already in complex with naïve and ligand-induced Trk receptors. In addition, a membrane sialidase mechanism initiated by NGF binding to TrkA has been indentified. It suggests a potentiation of GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live TrkA- and TrkB-expressing cells and primary neurons. These results establish a unique mode of regulation of Trk receptors by their natural ligand and define a new function for Neu1 sialidase. Preliminary data indicate that members of the family of tyrosine kinase receptors like epidermal growth factor receptor (EGFR) and insulin receptor are also under the same regulatory control of Neu1 sialidase. Recent reports from the laboratory have indicated that ligand-induced activation of the highly glycosylated Toll-like receptors, TLR-2,-3 and -4 is also dependent on Neu1 sialidase on the cell surface. Taken all together, the findings in this thesis uncover a Neu1 and MMP-9 cross-talk on the cell surface which is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and neuron function. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-26 11:44:51.418

RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815

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DAPK-1 is calcium-calmodulin regulated protein kinase involved in multiple cellular pathways including apoptosis, autophagy, cell survival and motility. The cytokine TNF-α has been reported to induce the degradation of DAPK-1. Here I identified the protease cathepsin B as a novel binding partner of DAPK-1 that protects DAPK-1 from TNF-α induced degradation. Using deletion mutants of DAPK-1, I mapped the cathepsin B binding domain on DAPK-1 to amino acids 836-947. Overexpression of this mini-protein DAPK-1(836-947) facilitated degradation of full-length DAPK-1 and apoptosis induced by TNFR-1. Moreover, siRNA mediated knock-down of DAPK-1 enhanced TNF-α induced apoptosis, confirming the role of DAPK-1 as a survival factor in the TNF-α signalling pathway. In addition, a splice variant of DAPK-1, which I have called s-DAPK-1, was discovered. s-DAPK-1 shares part of DAPK-1’s ankyrin repeats region and cytoskeletal binding domain, and possesses an unique tail region, which contains a cleavage site at its first two amino acids. Unlike DAPK-1, s-DAPK-1 does not contribute to apoptosis induced by high level of MEK/ERK signalling, but it does mimic DAPK-1’s function to induce membrane blebbing. The proteolytically processed form of s-DAPK-1 is more active in the induction of membrane blebbing, which may be due to its higher stability compared to that of full-length s-DAPK-1, suggesting that the tail region can control s-DAPK-1 stability and activity. Co-transfection of s-DAPK-1 and DAPK-1 leads to reduction in DAPK-1 expression level, suggesting a role for s-DAPK-1 to regulate DAPK-1 stability. The kinase domain of DAPK-1 is the region required for s-DAPK-1 to promote DAPK-1 degradation. Surprisingly, s-DAPK-1 does not bind directly to DAPK-1, suggesting that the interaction is indirect and mediated by as yet unidentified accessory proteins. Finally, the experiments with proteasome and lysosome inhibitors indicated that s-DAPK-1 induces DAPK-1 degradation via both lysosome and proteasome pathways.

In the current thesis we analyse protein phosphorylation pathways in P. falciparum, the protozoa responsible for the most virulent form of malaria in order to both understand the role and scope of this protein modification in the parasite, and to explore its feasibility as a new drug target. With the aim to map phosphorylation pathways controlled by P. falciparum Casein Kinase 2 (PfCK2), we developed a new chemical-biological approach based on γ-modified ATP analogues bearing reporting groups on the transferred phosphate in order to selectively tag CK2 substrates. Despite being able to efficiently synthesise a small set of analogues, the data presented here shows that the P-N linkage bond between the nucleotide and the tag is stable during the assay conditions but not during the product analysis due to its acidic liability (e.g. with HPLC, MALDI); suggesting that a different type of linkage should be chosen in the future. Detailed characterisation studies of the parasite PfCK2 presented here showed a number of important features differing from human CK2. Docking analyses with a CK2 inhibitor showed that the PfCK2 ATP binding pocket is smaller than human CK2 due to the presence of Val116 and Leu45 which in the human kinase are replaced by more bulky isoleucine residues: Ile120 and Ile49. The difference between the human and parasite CK2 orthologues extends further to mechanisms of activation and regulation. Shown here is the autophosphorylation of PfCK2 that, unlike the human orthologue, occurs within subdomain I at Thr63. This autophosphorylation is essential for full catalytic activity. In addition we also showed that Thr63 phosphorylation regulates the interaction between the calalytic α-subunit and the regulatory β2-subunit. Here, we also presented evidence for tyrosine phosphorylated proteins in parasite infected red blood cells. PfCK2 can act as a dual specificity kinase phosphorylating P. falciparum Minichromosome Maintenance protein 2 (PfMCM2) on Tyr16 in vitro. It is therefore possible that PfCK2 may contribute to tyrosine phosphorylation within the parasite. Finally, we also reported a study regarding MCM2-Ser13 phosphorylation which successfully identified PfCK1 as the kinase responsible for this event.

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.

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