Elucidation of regulatory mechanisms of the ITK and its substrate PLCγ1

This dissertation studies the regulatory mechanism of IL-2 inducible tyrosine kinase (ITK) and its substrate phospholipase C γ1 (PLCγ1). ITK and PLCγ1 are key mediators of the signaling pathway downstream of the T cell receptor that results in the T cell part of the adaptive immune response. In T cells, ITK is phosphorylated and activated by LCK. ITK then phosphorylates PLCγ1 at tyrosine 783 and activates phospholipase activity. This phosphorylation is dependent upon various coordinated intermolecular and intramolecular interactions within and between ITK and PLCγ1. This dissertation identifies and characterizes various regulatory interactions in ITK and its substrate PLCγ1 that regulates ITK mediated phosphorylation of PLCγ1. For ITK, this thesis explores the function of the N-terminal Pleckstrin homology (PH) domain in regulating the activation of the ITK. The ITK PH domain engages in direct interaction with ITK kinase domain and the region of the kinase domain interaction is mapped in the ITK PH domain. Mutations in ITK PH domain, that disrupts its interaction with kinase domain, lead to the increase in the activity of ITK. The ITK interaction surface mapped on the ITK PH domain lies adjacent to its phosphatidylinositol (3,4,5)-triphosphate (PI (3,4,5) P3) binding pocket. Hence, IP4, the soluble head group of (PI (3,4,5) P3) competes with the ITK kinase domain for ITK PH domain binding. Also, PI (3,4,5) P3 binding of ITK PH domain increases the catalytic activity of the ITK. In addition, PI (3,4,5) P3 binding increases the activation loop Y511 accessibility for LCK phosphorylation. This study expanded our knowledge on regulation of ITK by its N terminal PH domain (Chapter 3). ITK is a key modulator of immune response and has therefore been very attractive target for small molecule intervention for immunity related diseases such as autoimmune disease and asthma. The key novel regulatory ITK PH/kinase site can be a plausible allosteric target for small molecule discovery efforts. Part of this thesis contributes to assay development to detect the ITK PH/Kinase interaction in cells. Bimolecular Fluorescence complementation (BiFC) assay confirms that the ITK PH/Kinase interaction occurs in cell (Chapter 4). This assay can now be used to screen for small molecules that modulate the ITK PH/Kinase interaction. For PLCγ1, this thesis studies the mechanism of disruption of its autoinhibitory conformation. The PLCγ1 autoinhibitory conformation is characterized by an intramolecular interaction between the C terminal SRC homology 2 (SH2C) domain and the adjacent linker containing Y783. This conformation makes the crucial phosphorylation target (Y783) inaccessible to ITK. I have described the mechanism on how this autoinhibitory interaction is broken (Chapter 2). Our results suggest that the scaffold protein SLP-76 disrupts the autoinhibitory PLCγ1 conformation. More specifically, SLP-76 phosphotyrosine 173 (Y173) binds the SH2C domain of the PLCγ1, competing with the autoinhibitory conformation of PLCγ1 and releasing the linker, making the Y783 more accessible to ITK. Our results identify the new role of the scaffold protein SLP-76 adding to its previously described role in co-localizing the enzyme and substrate pair. This role is defined as the substrate priming as SLP-76 primes PLCγ1, the substrate for ITK for efficient phosphorylation. Our results provide further understanding of the regulation of ITK mediated phosphorylation of PLCγ1 and create a foundation for small molecule discovery efforts that should yield new ways to either enhance or diminish T cell function

Machine learning approach to detect Land Use Land Cover (LULC) change in Chure region of Sarlahi district, Nepal

Land Use and Land Cover (LULC) changes are major global environmental issues, affecting ecological systems, climate change, and biodiversity. The Chure region of the Sarlahi district in Nepal is a critical ecological zone that has experienced significant LULC changes in recent years. In this study, our aim was to apply a machine learning approach to detect LULC changes in the Chure region using Google Earth Engine (GEE) and the Random Forest classifier. We utilized Landsat imagery of 2007 and 2022 to generate land cover maps for each year, which were then compared to identify changes over time. The major findings of this study indicate that the forest cover in the region has increased by approximately 16% over the past 15 years, while the agriculture and built-up areas have also shown a significant increase. Conversely, the barren land and water areas have decreased. The classifier obtained an overall accuracy of 85.7% and a kappa coefficient of 81.2% for the year 2022, and an overall accuracy of 82.2% and a kappa coefficient of 76.8% for the year 2007, which demonstrates the high accuracy of the proposed approach. The use of GEE and random forest classifiers provided a cost-effective and efficient method for analysing large datasets and producing accurate LULC maps. Our findings can inform policymakers and conservationists about the need for sustainable land management practices to preserve the ecological integrity of the Chure region. The approach can be applied to other regions to monitor and manage LULC changes and support effective conservation efforts

Familial Alzheimer’s disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues

Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated

Effects of presenilin-1 familial Alzheimer’s disease mutations on γ-secretase activation for cleavage of amyloid precursor protein

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase which cleaves within the transmembrane domain of over 150 peptide substrates. Dominant missense mutations in PS1 cause early-onset familial Alzheimer’s disease (FAD); however, the exact pathogenic mechanism remains unknown. Here we combined Gaussian accelerated molecular dynamics (GaMD) simulations and biochemical experiments to determine the effects of six representative PS1 FAD mutations (P117L, I143T, L166P, G384A, L435F, and L286V) on the enzyme-substrate interactions between γ-secretase and amyloid precursor protein (APP). Biochemical experiments showed that all six PS1 FAD mutations rendered γ-secretase less active for the endoproteolytic (ε) cleavage of APP. Distinct low-energy conformational states were identified from the free energy profiles of wildtype and PS1 FAD-mutant γ-secretase. The P117L and L286V FAD mutants could still sample the “Active” state for substrate cleavage, but with noticeably reduced conformational space compared with the wildtype. The other mutants hardly visited the “Active” state. The PS1 FAD mutants were found to reduce γ-secretase proteolytic activity by hindering APP residue L49 from proper orientation in the active site and/or disrupting the distance between the catalytic aspartates. Therefore, our findings provide mechanistic insights into how PS1 FAD mutations affect structural dynamics and enzyme-substrate interactions of γ-secretase and APP

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society (JACS), copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/jacs.9b13405.γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembranemimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.NIH R01 grant GM 122894NIH grant P30GM110761NIH grant P41GM11113

Elucidation of regulatory mechanisms of the ITK and its substrate PLCγ1

This dissertation studies the regulatory mechanism of IL-2 inducible tyrosine kinase (ITK) and its substrate phospholipase C γ1 (PLCγ1). ITK and PLCγ1 are key mediators of the signaling pathway downstream of the T cell receptor that results in the T cell part of the adaptive immune response. In T cells, ITK is phosphorylated and activated by LCK. ITK then phosphorylates PLCγ1 at tyrosine 783 and activates phospholipase activity. This phosphorylation is dependent upon various coordinated intermolecular and intramolecular interactions within and between ITK and PLCγ1. This dissertation identifies and characterizes various regulatory interactions in ITK and its substrate PLCγ1 that regulates ITK mediated phosphorylation of PLCγ1. For ITK, this thesis explores the function of the N-terminal Pleckstrin homology (PH) domain in regulating the activation of the ITK. The ITK PH domain engages in direct interaction with ITK kinase domain and the region of the kinase domain interaction is mapped in the ITK PH domain. Mutations in ITK PH domain, that disrupts its interaction with kinase domain, lead to the increase in the activity of ITK. The ITK interaction surface mapped on the ITK PH domain lies adjacent to its phosphatidylinositol (3,4,5)-triphosphate (PI (3,4,5) P3) binding pocket. Hence, IP4, the soluble head group of (PI (3,4,5) P3) competes with the ITK kinase domain for ITK PH domain binding. Also, PI (3,4,5) P3 binding of ITK PH domain increases the catalytic activity of the ITK. In addition, PI (3,4,5) P3 binding increases the activation loop Y511 accessibility for LCK phosphorylation. This study expanded our knowledge on regulation of ITK by its N terminal PH domain (Chapter 3). ITK is a key modulator of immune response and has therefore been very attractive target for small molecule intervention for immunity related diseases such as autoimmune disease and asthma. The key novel regulatory ITK PH/kinase site can be a plausible allosteric target for small molecule discovery efforts. Part of this thesis contributes to assay development to detect the ITK PH/Kinase interaction in cells. Bimolecular Fluorescence complementation (BiFC) assay confirms that the ITK PH/Kinase interaction occurs in cell (Chapter 4). This assay can now be used to screen for small molecules that modulate the ITK PH/Kinase interaction. For PLCγ1, this thesis studies the mechanism of disruption of its autoinhibitory conformation. The PLCγ1 autoinhibitory conformation is characterized by an intramolecular interaction between the C terminal SRC homology 2 (SH2C) domain and the adjacent linker containing Y783. This conformation makes the crucial phosphorylation target (Y783) inaccessible to ITK. I have described the mechanism on how this autoinhibitory interaction is broken (Chapter 2). Our results suggest that the scaffold protein SLP-76 disrupts the autoinhibitory PLCγ1 conformation. More specifically, SLP-76 phosphotyrosine 173 (Y173) binds the SH2C domain of the PLCγ1, competing with the autoinhibitory conformation of PLCγ1 and releasing the linker, making the Y783 more accessible to ITK. Our results identify the new role of the scaffold protein SLP-76 adding to its previously described role in co-localizing the enzyme and substrate pair. This role is defined as the substrate priming as SLP-76 primes PLCγ1, the substrate for ITK for efficient phosphorylation. Our results provide further understanding of the regulation of ITK mediated phosphorylation of PLCγ1 and create a foundation for small molecule discovery efforts that should yield new ways to either enhance or diminish T cell function.</p

Accessible and usable web interface forpPower grid database

Different stakeholders of the electric power grid sector need data for their research and decision-making tasks. However, the power grid field lacks an accessible and easy-to-use system that contains openly available data sources that is open to use for everyone. This thesis creates a prototype of an accessible and usable web interface for the power grid database by following the user-centered design (UCD) methodology. The web interface is targeted to be used by different stakeholders of the power grid sector. The principles of universal design and concepts of accessibility and usability were considered throughout different activities in the thesis. Different qualitative research methods and research methods from human-computer interaction (HCI) have been used in this study. The research methods employed in this thesis are survey, coding, interviews, paper prototyping, personas, automatic testing, heuristic testing, and discounted testing. Further, ethnographic observation and thinking out aloud methods have also been used. The prototype development process followed an iterative approach for finding user requirements and deliver a prototype of a web-based interface for the power grid database. The prototype was tested for its accessibility and usability. The prototype presented uses modern web technologies and is found to meet the accessibility and usability requirements of its target users. The prototype and the instructions to install it have also been uploaded along with this thesis. Finally, some topics for future work to further extend this study and to make the software prototype more effective have been presented

Scaffold Protein SLP-76 Primes PLCγ1 for Activation by ITK-Mediated Phosphorylation

Activation of the phospholipase, PLCγ1, is critical for proper T cell signaling following antigen receptor engagement. In T cells, the Tec family kinase, ITK, phosphorylates PLCγ1 at tyrosine 783 (Y783) leading to activation of phospholipase function and subsequent production of the second messengers IP3 and DAG. In this work we demonstrate that PLCγ1 can be primed for ITK mediated phosphorylation on Y783 by a specific region of the adaptor protein, SLP-76. The SLP-76 phosphotyrosine containing sequence, pY173IDR, does not conform to the canonical recognition motif for an SH2 domain yet binds with significant affinity to the C-terminal SH2 domain of PLCγ1 (SH2C). The SLP-76 pY173 motif competes with the autoinhibited conformation surrounding the SH2C domain of PLCγ1 leading to exposure of the ITK recognition element on the PLCγ1 SH2 domain and release of the target tyrosine, Y783. These data contribute to the evolving model for the molecular events occurring early in the T cell activation process.This is a manuscript of an article published as Devkota, Sujan, Raji E. Joseph, Lie Min, D. Bruce Fulton, and Amy H. Andreotti. "Scaffold protein SLP-76 primes PLCγ1 for activation by ITK-mediated phosphorylation." Journal of molecular biology 427, no. 17 (2015): 2734-2747. doi: 10.1016/j.jmb.2015.04.012. Posted with permission.</p

Profiles and Inhospital Outcomes of Patients with Acute Upper Gastrointestinal Bleeding in a Tertiary Care Center

Background: Acute upper gastrointestinal bleeding is one of the common and life threatening condition presenting in emergency. The aim of the present study was to determine the clinical profile and in-hospital outcomes in these patients admitted at a tertiary care center in Kathmandu, Nepal. Materials and Methods: This is a descriptive, observational study conducted over 9 months (October 2021– July 2022). All consecutive patients aged 16 years and above admitted in the hospital ward with the history of acute bleeding were included in the study after informed consent. Demographic data, clinical, laboratory and endoscopic data were noted during the hospital stay. Statistical analysis was done used SPSS v.24. Results: The mean age of our study population (N = 132) was 46.92 years. Among 132 patients who underwent endoscopy, 43.9% had portal hypertension related bleeding, 41.7% had ulcer related bleeding, 5.3% had malignancies, 3% had corrosive intake. No etiology was found in 3 patients. Hematemesis with melena was the most common mode (53.8%) of presentation to the hospital.Shock was preset in 25% of patients at presentation to the hospital. In-hospital re-bleeding rate and mortality were 12.7% and 9.8% respectively. Conclusion: Portal hypertension related bleeding followed by ulcers related bleeding is the common cause of acute upper gastrointestinal bleeding. In-hospital mortality was 9.8%

Decline in Honeybees and Its Consequences for Beekeepers and Crop Pollination in Western Nepal

In understudied regions of the world, beekeeper records can provide valuable insights into changes in pollinator population trends. We conducted a questionnaire survey of 116 beekeepers in a mountainous area of Western Nepal, where the native honeybee Apis cerana cerana is kept as a managed bee. We complemented the survey with field data on insect–crop visitation, a household income survey, and an interview with a local lead beekeeper. In total, 76% of beekeepers reported declines in honeybees, while 86% and 78% reported declines in honey yield and number of beehives, respectively. Honey yield per hive fell by 50% between 2012 and 2022, whilst the number of occupied hives decreased by 44%. Beekeepers ranked climate change and declining flower abundance as the most important drivers of the decline. This raises concern for the future food and economic security of this region, where honey sales contribute to 16% of total household income, and where Apis cerana cerana plays a major role in crop pollination, contributing more than 50% of all flower visits to apple, cucumber, and pumpkin. To mitigate further declines, we promote native habitat and wildflower preservation, and using well-insulated log hives to buffer bees against the increasingly extreme temperature fluctuations