CONTRIBUTION OF A SPERM PROTEIN, PAWP, TO THE SIGNAL TRANSDUCT PATHWAY DURING VERTEBRATE FERTILIZATION

PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early and later egg activation events including meiotic resumption, pronuclear formation and egg cleavage. Based on the fact that calcium increase is universally accepted as the sole requirement for egg activation, we hypothesized that PAWP is an upstream regulator of the calcium signaling pathway during fertilization. Intracellular calcium increase was detected by two-photon laser scanning fluorescence microscopy following microinjection of recombinant PAWP into Xenopus oocytes, bolstering our hypothesis and suggesting the involvement of a novel PAWP-mediated signaling pathway during fertilization. The N-terminal of PAWP shares a high homology to WW domain binding protein while the C-terminal half contains a functional PPXY motif, which allows it to interact with group I WW domain proteins. These structural considerations together with published data indicating that PPXY synthetic peptide derived from PAWP inhibits ICSI-induced fertilization led to the hypothesis that PAWP triggers egg activation by binding to a group I WW domain protein in the oocyte. By far-Western analysis of oocyte cytoplasmic fraction, PAWP was found to bind to a 52 kDa protein. The competitive inhibition studies with PPXY synthetic peptide, WW domain constructs, and their point mutants demonstrated that the interaction between PAWP and its binding partner is specifically via the PPXY-WW domain module. The 52 kDa protein band crossreacted with antibodies against group I WW domain protein YAP in Western blot assay, indicating that this 52 kDa PAWP binding partner is either YAP or a YAP-related protein. In addition, the far-Western competitive inhibition studies with recombinant GST fusion protein YAP and another WW domain-containing protein, TAZ, demonstrated that the binding of PAWP to its binding partner was significantly reduced by TAZ, providing evidence that TAZ could be the 52 kDa protein candidate. Mass spectrometry was employed to identify this PAWP binding partner candidate. However, due to the low abundance of the candidate protein and the complexity of the sample, several strategies are still needed to enrich this protein. This study correlates PAWP induced meiotic resumption and calcium efflux at fertilization and uncovers a 52 kDa candidate WW domain protein in the oocyte cytoplasm that most likely interacts with PAWP to trigger egg activation.

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 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.

HOME-BASED REHABILITATION AND ITS IMPACT ON HOSPITAL UTILIZATION

There is compelling evidence for the effectiveness of home-based occupational therapy and physiotherapy rehabilitation for community dwelling elderly who may struggle with basic activities and the functions of daily living and mobility. Nonetheless, an estimated 2% of home care’s elderly clients receive these therapies. Ontario’s home care data indicates that 78% of clients that could benefit from these specific therapies are not receiving them. The study examined a subset of elderly clients receiving home care following a hospital discharge during 2009-2010. The aim of this study was to: understand the difference between those home care clients who received occupational therapy or physiotherapy and those who did not; and determine if receiving these therapies impacted the utilization of hospital emergency departments and inpatient admissions. A retrospective cohort design and multivariate and survival analysis of hospital and home care administrative data structured the study. Results suggest that home-based rehabilitation is offered to a minority of the home care population. Distinct client characteristics and process variables significantly associated with the increased likelihood of receiving home-based occupational and physical therapies included: clients who were older, females, admitted to home care from hospital inpatient units, assessed as non-acute for clinical and service needs and required more home making support and assistance with activities of daily living. Almost one quarter of the total sample returned to hospital. Visits to emergency departments accounted for the greater part of hospital utilization and primarily for sub-acute general symptoms and signs, post-procedural complications, infections or acute episodes from chronic obstructive pulmonary disease and renal failure. Slightly over half of the clients returning to hospital did not receive home-based rehabilitation. Clients who received occupational therapy returned to the hospital sooner following their home care admission whereas clients receiving physiotherapy spent the longest time before rehospitalizing. The majority of the clients receiving occupational therapy were admitted to home care having just resolved sub-acute conditions or symptoms, many of which are known to influence functional and physical decline. Moreover, analysis of process variables indicated that the wait time for a referral to occupational therapy was two times longer compared to physiotherapy. These same clients also waited, on average, over one month before an occupational therapist’s first visit. The need to discriminate who receives home-based rehabilitation is essential to understanding how specific therapies contribute to improving systems outcomes. This study is the first examination that focuses specifically on home-based occupational therapy and physiotherapy rehabilitation and the client characteristics and process variables associated with receiving/not receiving these therapies and the impact these factors have on the time-to-rehospitalization.

A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.