Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Differential contributions of the mIPS and PMd in the movement planning of reaches investigated through HD-tDCS

To reach for a target, we must formulate a movement plan - a difference vector of the target position with respect to the starting hand position. While it is known that the medial part of the intraparietal sulcus (mIPS) and the dorsal premotor (PMd) activity reflects aspects of a kinematic plan for a reaching movement, it is unclear whether or how the two regions may differ. We investigated the functional roles of the mIPS and PMd in the planning of reaching movements using high definition transcranial direct current stimulation (HD-tDCS) and examined changes in horizontal endpoint error when participants were subjected to anodal and cathodal stimulation. The left mIPS and PMd were functionally localized with fMRI in each participant using an interleaved center-out pointing and saccade task and mapped onto the scalp using Brainsight. We adopted a randomized, single-blind design and applied anodal and cathodal stimulation (2mA for 20 min; 3cm radius 4x1 electrode placement) during 4 separate visits scheduled at least a week apart. Each participant performed 250 baseline, stimulation, and post-stimulation memory-guided reaches starting from one of two initial hand positions (IHPs) to one of 4 briefly flashed targets (20 cm distant, 5 cm apart horizontally) while fixating on a straight-ahead cross located at the target line. Separate 2-way repeated measures ANOVAs of horizontal endpoint error difference after cathodal tDCS at each stimulation site revealed a significant IHP by target position interaction effect at the left mIPS, and significant IHP and target main effects at the left PMd. Behaviorally, these effects corresponded to IHP-dependent contractions after cathodal mIPS tDCS and IHP-independent contractions after cathodal PMd tDCS. These results suggest that the movement vector is not yet formed at the input level of mIPS, but is encoded at the input of PMd. These results also indicate that tDCS is a viable, useful method in investigating movement planning properties through temporary perturbations of the system.

Glycemic State Regulates Brain-Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventricular Nucleus of the Hypothalamus

Brain derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and binds to the tropomyosin-related kinase B (TrkB) receptor. Like other neurotrophic factors, BDNF is involved in the development and differentiation of neurons. Recently, studies have suggested important roles for BDNF in the regulation of energy homeostasis. The paraventricular nucleus (PVN) is critical for normal energy balance contains high levels of both BDNF and TrkB mRNA. Studies have shown that microinjections of BDNF into the PVN increase energy expenditure, suggesting BDNF plays a role in energy homeostasis through direct actions in this hypothalamic nucleus. We used male Sprague-Dawley rats to perform whole-cell current-clamp experiments from PVN neurons in slice preparation. BDNF was bath applied at a concentration of 2nM and caused depolarizations in 54% of neurons (n = 25; mean change in membrane potential: 8.9 ± 1.2 mV), hyperpolarizations in 23% (n = 11; mean change in membrane potential: -6.7 ± 1.4 mV), while the remaining cells tested were unaffected. Previous studies showing effects of BDNF on γ-aminobutyric acid type A (GABAA) mediated neurotransmission in PVN led us to examine if these BDNF-mediated changes in membrane potential were maintained in the presence of tetrodotoxin (TTX) sodium channel blocker (N = 9; 56% depolarized, 22% hyperpolarized, 22% non-responders) and bicuculline (GABAA antagonist) (N = 12; 42% depolarized, 17% hyperpolarized, 41% non-responders), supporting the conclusion that these effects on membrane potential were postsynaptic. We also evaluated the effects of BDNF on these neurons across varying physiologically relevant extracellular glucose concentrations. At 10 mM 23% (n = 11; mean: -6.7 ± 1.4 mV) of PVN neurons hyperpolarized in response to BDNF treatment, whereas at 0.2 mM glucose, 71% showed hyperpolarizing effects (n = 12; mean: -6.3 ± 2.8 mV). Our findings reveal that BDNF has direct impacts on PVN neurons and that these neurons are capable of integrating multiple sources of metabolically relevant input. Our analysis regarding glucose concentrations and their effects on these neurons’ response to other metabolic signals emphasizes the importance of using physiologically relevant conditions for study of central pathways involved in the regulation of energy homeostasis.

(Re)Imagining Relationality: Brad Isaacs and The Map of the Empire

The exhibition, The Map of the Empire (30 March – 6 May, 2016), featured photography, video, and installation works by Toronto-based artist, Brad Isaacs (Mohawk | mixed heritage). The majority of the artworks within the exhibition were produced from the Canadian Museum of Nature’s research and collections facility (Gatineau, Québec). The Canadian Museum of Nature (CMN), is the national natural history museum of (what is now called) Canada, with its galleries located in Ottawa, Ontario. The exhibition was the first to open at the Centre for Indigenous Research Creation at Queen’s University under the supervision of Dr. Dylan Robinson. Through the installment of The Map of the Empire, Isaacs effectively claimed space on campus grounds – within the geopolitical space of Katarokwi | Kingston – and pushed back against settler colonial imaginings of natural history. The Map of the Empire explored the capacity of Brad’s artistic practice in challenging the general belief under which natural history museums operate: that the experience of collecting/witnessing/interacting with a deceased and curated more-than-human animal will increase conservation awareness and facilitate human care towards nature. The exhibition also featured original poetry by Cecily Nicholson, author of Triage (2011) and From the Poplars (2014), as a response to Brad’s artwork. I locate the work of The Map of the Empire within the broader context of curatorship as a political practice engaging with conceptual and actualized forms of slow violence, both inside of and beyond the museum space. By unmapping the structures of slow, showcased and archived violence within the natural history museum, we can begin to radically transform and reimagine our connections with more-than-humans and encourage these relations to be reciprocal rather than hyper-curated or preserved.

Functional characterization of a Kar3/Vik1-like Kinesin-14 heterodimer from the filamentous multinucleate fungus Ashbya gossypii

Kinesins are motor proteins that convert chemical energy from ATP hydrolysis into mechanical energy used to generate force along microtubules, transporting organelles, vesicles, and proteins within the cell. Kar3 kinesins are microtubule minus-end-directed motors with pleiotropic functions in mating and mitosis of budding and fission yeast. In Saccharomyces cerevisiae, Kar3 is multifunctionalized by two non-catalytic companion proteins, Vik1 and Cik1. A Kar3-like kinesin and a single Vik1/Cik1 ortholog are also expressed by the filamentous fungus Ashbya gossypii, which exhibits different nuclear movement challenges and unique microtubule dynamics from its yeast relatives. We hypothesized that these differences in A. gossypii physiology could translate into interesting and novel differences in its versions of Kar3 and Vik1/Cik1. Presented here is a structural and functional analysis of recombinantly expressed and purified forms of these motor proteins. Compared to the previously published S. cerevisiae Kar3 motor domain structure (ScKar3MD), AgKar3MD displays differences in the conformation of the ATPase pocket. Perhaps it is not surprising then that we observed the maximal microtubule-stimulated ATPase rate (kcat) of AgKar3MD to be approximately 3-fold slower than ScKar3MD, and that the affinity of AgKar3MD for microtubules (Kd,MT) was lower than ScKar3MD. This may suggest that elements that compose the ATPase pocket and that participate in conformational changes required for efficient ATP hydrolysis or products release work differently for AgKar3 and ScKar3. There are also subtle structural differences in the disposition of the secondary structural elements in the small lobe (B1a, B1b, and B1c) at the edge of the motor domain of AgKar3 that may reflect the enhanced microtubule-depolymerization activity that we observed for this motor, or they could relate to its interactions with a different regulatory companion protein than its budding yeast counterpart. Although we were unable to gain experimentally determined high-resolution information of AgVik1, the results of Phyre2-based bioinformatics analyses may provide a structural explanation for the limited microtubule-binding activity we observed. These and other fundamental differences in AgKar3/Vik1 could explain divergent functionalities from the ScKar3/Vik1 and ScKar3/Cik1 motor assemblies.

Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.

Functional Characterization of TAFI mutants Resistant to Activation by Thrombin, Thrombin-Thrombomodulin or Plasmin

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo.

Structural and Functional Characterization of a Novel Heterodimeric Kinesin in Candida albicans

Kinesins are molecular motors that transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the MT cytoskeleton. Their force-generating functions arise from conformational changes in their motor domain as ATP is bound and hydrolyzed, and products are released. In the budding yeast Saccharomyces cerevisiae, the Kar3 kinesin forms heterodimers with one of two non-catalytic kinesin-like proteins, Cik1 and Vik1, which lack the ability to bind ATP, and yet they retain the capacity to bind MTs. Cik1 and Vik1 also influence and respond to the MT-binding and nucleotide states of Kar3, and differentially regulate the functions of Kar3 during yeast mating and mitosis. The mechanism by which Kar3/Cik1 and Kar3/Vik1 dimers operate remains unknown, but has important implications for understanding mechanical coordination between subunits of motor complexes that traverse cytoskeletal tracks. In this study, we show that the opportunistic human fungal pathogen Candida albicans (Ca) harbors a single version of this unique form of heterodimeric kinesin and we present the first in vitro characterization of this motor. Like its budding yeast counterpart, the Vik1-like subunit binds directly to MTs and strengthens the MT-binding affinity of the heterodimer. However, in contrast to ScKar3/Cik1 and ScKar3/Vik1, CaKar3/Vik1 exhibits weaker overall MT-binding affinity and lower ATPase activity. Preliminary investigations using a multiple motor motility assay indicate CaKar3/Vik1 may not be motile. Using a maltose binding protein tagging system, we determined the X-ray crystal structure of the CaKar3 motor domain and observed notable differences in its nucleotide-binding pocket relative to ScKar3 that appear to represent a previously unobserved state of the active site. Together, these studies broaden our knowledge of novel kinesin motor assemblies and shed new light on structurally dynamic regions of Kar3/Vik1-like motor complexes that help mediate mechanical coordination of its subunits.

Functional and Physiological Determinants of Perceived Disability in Individuals Diagnosed with Osteoarthritis of the Hip

Objectives: The main objective of this pilot study was to investigate which standardized functional and physiological test best predicted perceived disability in a single group of 21 individuals diagnosed with osteoarthritis of the hip. Design: Men and women between 60 and 70 years old with osteoarthritis of the hip were selected. If participants passed study criteria, the Western Ontario McMaster University questionnaire (WOMAC), 6 Minute Walk Test (6MWT) and Timed up and Go (TUG), strength testing and aerobic testing were obtained in one single assessment. Results: Regression analysis revealed that wait time, hip abduction strength of the affected side, Aerobic Capacity (VO2 Peak), hip Extension Peak Torque, hip Flexion Peak Torque, TUG and 6MWT were significantly correlated with the WOMAC. Yet, the 6MWT had the highest significant correlation (r = -0.86, p ≤ 0.0001); R2 = 0.75 or 75% with the WOMAC total scores, (r = -0.82, p ≤ 0.0001); R2 = 0.67 or 67% with the WOMAC function and (r = -0.60, p = .002); R2 = 0.36 or 36% with the WOMAC stiffness. While the VO2 Peak revealed the highest significant correlation (r = 0.76, p ≤ .0001); R2 = 0.57 or 57% with the WOMAC pain. Conclusions: The 6MWT and the VO2 Peak seem to be essential functional and physiological assessment tools to determine perceived disability in individuals with hip OA. The perceived disability may provide new or comprehensive knowledge of the disability problems experienced by individuals with osteoarthritis of the hip, and the association of patient perception with objective measures of functional and physiological capacity might strengthen the clinical value of this knowledge.