Reframing viral infections as acute metabolic disorders: dengue viruses and their dependency on host metabolic pathways

2022 Summer.Includes bibliographical references.Dengue viruses (DENVs) are the etiological agent of the world's most aggressive arthropod-borne disease. At present, there are no available antivirals against DENVs. This fact underscores a dire need to examine host-virus interactions to identify and develop novel therapeutic approaches. As obligate intracellular parasites, DENVs are reliant upon and hijack several host metabolic pathways both to fulfill their replicative needs, and to evade the host immune response. We and others have previously established that infection with DENVs causes significant perturbation to host lipid metabolism, including elevations in sphingolipids in both the human and mosquito host. In addition, we and others previously discovered that the DENV NS1 protein increases sialidase activity in both in vitro and in vivo models leading to increased endothelial hyperpermeability and vascular leakage which are hallmarks of severe dengue. To further clarify and characterize these previous works, we have performed siRNA-mediated loss of function studies using human hepatoma cells (Huh7 cells) on several metabolic pathways altered during DENV2 infection. First, we examined the role of acyl-CoA thioesterases, enzymes responsible for controlling the intracellular balance of activated fatty acids and free fatty acids, on the DENV2 lifecycle. In these analyses, we determined that the cytosolic ACOT1 enzyme had an inhibitory effect on DENV2 replication and release, while mitochondrial ACOT (ACOTs 2 and 7) functionality was critical for viral replication and release. Moreover, we identified several enzymes within the ACOT family whose expression was dependent on ACOT2 and ACOT7 expression. These results highlighted complex relationships between ACOTs and DENVs, as well as identified yet unknown functional interdependence between ACOT enzymes. Next, we expanded our previous understanding of the relationship between DENVs and the human sialidase enzymes (NEU1-4). While previously studies linked upregulation of these enzymes with DENV2 pathology, we provide the first evidence showing that NEU1-4 functionality is vital for DENV2 genome replication and viral egress. Moreover, our analyses also revealed previously unknown functionality of NEU4 or its downstream products as transcriptional regulators for NEU1-3. Finally, we provide the first profile of the effect of loss of function of enzymes within the entire sphingolipid metabolic pathway (as identified through KEGG pathway database) on the DENV2 life cycle. In this study, we identified that enzymes involved the sphingomyelinase and salvage pathways of ceramide synthesis as opposed to de novo ceramide synthesis were critical to DENV2 release from Huh7 cells. In addition, we determined that enzymes involved in the synthesis and degradation of glycosphingolipids were vital for DENV2 release. An especially intriguing result within this arm of sphingolipid metabolism was that the two enzymes which hydrolyze GluCer had differential effects on DENV2 replication and release. GBA1 (lysosomal) had an antiviral effect on DENV2, while GBA2 (non-lysosomal) was required for DENV2 replication and release. This prompted us to profile the changes that occur to glycosphingolipids (GSLs) during infection, and we uncovered several species of GSLs that are elevated during infection. Moreover, we identified that Ambroxol HCl, a pharmaceutical GBA1 chaperone/GBA2 inhibitor, was able to abrogate these elevations in GSLs. Combined, our results allowed us to propose a novel function for GBA2 as a GluCer recycling enzyme during DENV2 infection. In conclusion, together, the work in this dissertation highlights critical metabolic nodes that impact virus replication and provides new directions for investigating viral infections as acute metabolic diseases

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Functions and mechanisms of Rab46 in endothelial cells

Endothelial cells maintain vascular integrity by regulating a number of physiological and pathophysiological processes, including haemostasis, thrombosis and inflammation. A pivotal contribution to these processes is the exocytosis of cargo from specialized endothelial storage organelles, namely Weibel-Palade bodies (WPBs). WPBs provide an intracellular storage pool of pro-thrombotic and pro-inflammatory mediators which can be differentially released in response to different stimuli. Ca2+ raising agonists such as thrombin and histamine, respectively released following vascular injury or an immunogenic insult, evoke WPB exocytosis. However, inappropriate and untimely exocytosis of WPBs can promote the pro-thrombotic and pro-inflammatory environment evident in cardiovascular diseases. The mechanisms underlying differential cargo release in order to produce physiologically distinct responses are poorly understood. Some Rab GTPase family members, have been reported to be implicated in regulating the exocytosis of WPBs. Here, we describe a novel Rab GTPase (Rab46) in endothelial cells that is located on WPBs. Super-resolution microscopy confirmed that Rab46 is juxtaposed to von Willebrand Factor (vWF), on the cytosolic side of individual WPBs, whilst quantitative imaging analysis suggested that Rab46 may regulate a subpopulation of WPBs. Interestingly, Rab46 was necessary for acute histamine, but not thrombin, WPB trafficking towards the perinuclear area identified as the Microtubule Organizing Centre (MTOC). Biochemical analysis and mass spectrometry was used to investigate the molecular mechanisms underlying Rab46-dependent retrograde trafficking and the dynein heavy chain was identified as a candidate effector protein. Further biochemical experiments suggested a direct interaction between endogenous Rab46 and the dynein motor complex. Taken together, these results suggest that after acute histamine stimulation, dynein-bound Rab46 mediates retrograde transport of a subset of WPBs along microtubules to the MTOC. These observations indicate Rab46 as a key regulator of differential WPB cargo secretion, allowing an appropriate acute pro-inflammatory response whilst avoiding release of excessive pro-thrombotic mediators. Characterization of in vivo model of Rab46 represents the beginning of understanding the physiological contribution of Rab46 as well as its response to pathological conditions. Understanding the Rab46/WPB signalling axis, both in vitro and in vivo, could be important for achieving better appreciation of how the endothelial cell fine-tunes it’s secretory response and thereby providing novel therapeutic targets for the prevention of endothelial dysfunction, which is often the trigger for cardiovascular diseases

Application of multivariate statistics and machine learning to phenotypic imaging and chemical high-content data

Image-based high-content screens (HCS) hold tremendous promise for cell-based phenotypic screens. Challenges related to HCS include not only storage and management of data, but critical analysis of the complex image-based data. I implemented a data storage and screen management framework and developed approaches for data analysis of a number high-content microscopy screen formats. I visualized and analysed pilot screens to develop a robust multi-parametric assay for the identification of genes involved in DNA damage repair in HeLa cells. Further, I developed and implemented new approaches for image processing and screen data normalization. My analyses revealed that the ubiquitin ligase RNF8 plays a central role in DNA-damage response and that a related ubiquitin ligase RNF168 causes the cellular and developmental phenotypes characteristic for the RIDDLE syndrome. My approaches also uncovered a role for the MMS22LTONSL complex in DSB repair and its role in the recombination-dependent repair of stalled or collapsed replication forks. The discovery of novel bioactive molecules is a challenge because the fraction of active candidate molecules is usually small and confounded by noise in experimental readouts. Cheminformatics can improve robustness of chemical high-throughput screens and functional genomics data sets by taking structure-activity relationships into account. I applied statistics, machine learning and cheminformatics to different data sets to discern novel bioactive compounds. I showed that phenothiazines and apomorphines are regulators for cell differentiation in murine embryonic stem cells. Further, I pioneered computational methods for the identification of structural features that influence the degradation and retention of compounds in the nematode C. elegans. I used chemoinformatics to assemble a comprehensive screening library of previously approved drugs for redeployment in new bioassays. A combination of chemical genetic interactions, cheminformatics and machine learning allowed me to predict novel synergistic antifungal small molecule combinations from sensitized screens with the drug library. In another study on the biological effects of commonly prescribed psychoactive compounds, I discovered a strong link between lipophilicity and bioactivity of compounds in yeast and unexpected off-target effects that could account for unwanted side effects in humans. I also investigated structure-activity relationships and assessed the chemical diversity of a compound collection that was used to probe chemical-genetic interactions in yeast. Finally, I have made these methods and tools available to the scientific community, including an open source software package called MolClass that allows researchers to make predictions about bioactivity of small molecules based on their chemical structure

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Molecular mechanisms and targets of new anticancer treatments

The work presented in this thesis is an effort to decipher and understand the mechanism of action (MOA) of anticancer agents by building on and complementing chemical proteomics methods. The backbone of the thesis relies on a recent method called Functional Identification of Target by Expression Proteomics (FITExP) developed in Zubarev lab, where drug induced proteomic signatures are analyzed in various cell lines and top differentially regulated proteins with consistent behavior are determined, among which the drug target and mechanistic proteins are usually present. FITExP relies on the assumption that proteins most affected with a perturbation have a higher probability of being involved in that process. In this regard, Paper I aimed to enhance the performance of FITExP analysis by merging proteomic data from drug-treated matrix attached and detached cells. This is while the majority if not all proteomics and molecular biology experiments are performed in matrix attached cells, as the general belief is that detached cells lose their structural integrity and do not harbor valuable information. However, detached cells are those that are more sensitive to chemotherapeutics and might reflect the proteome changes better. The comparative proteomics of living and dying cells improved FITExP performance with regards to identification of targets and provided insight about proteins involved in cellular life and death decisions. Furthermore, the orthogonal partial least squares-discriminant analysis (OPLS-DA) paradigm presented in this study, was used throughout the thesis for contrasting and visualizing the proteomic signature of a molecule against others, to reveal targets and specific proteins changing in response to the molecule of interest. In Paper II, as a further development of FITExP and to demonstrate its applicability in a broader context, we built a proteome signature library of 56 clinical and experimental anticancer agents in A549 lung adenocarcinoma cell line. This resource called ProTargetMiner can be used for different purposes. The proximity of compounds in hierarchical clustering or t-SNE could be used for prediction of the mechanism of new compounds. Contrasting each molecule against other treatments using the OPLS-DA scheme presented in Paper I, revealed drug targets, mechanistic proteins, resistance factors, drug metabolizing enzymes and effects on protein complexes. Representative examples were used to demonstrate that the specificity factors extracted from the OPLS-DA models can help identify subtle but biologically significant processes, even when such an effect is as low as 15% fold change. Furthermore, we showed that the inclusion of 8-10 contrasting molecules in the OPLS-DA models can produce enough specificity for drug target deconvolution, which offered a miniaturization opportunity. Therefore, we built three deeper datasets using 9 compounds that showed the most diverse proteome changes in the orthogonal space in three cell lines from major cancer types: A549 lung, MCF-7 breast and RKO colon cancers. These datasets provide a unique depth of 7398, 8735 and 8551 respectively, with no missing values. Subsequently, a Shiny package was created in R, which can employ these datasets as a resource and merge it with user data and provide OPLS-DA output and target deconvolution opportunity for new compounds. Finally, using the original ProTargetMiner data, we also built a first of its kind proteomic correlation database which can find applications in deciphering the function of uncharacterized proteins. Moreover, the resource helped to identify a set of core or untouchable proteins with stable expression across all the treatments, revealing essential functions within the cells. Such proteins could be used as house-keeping controls in molecular biology experiments. In paper III, we combined FITExP with other chemical proteomics tools Thermal Proteome Profiling (TPP) and multiplexed redox proteomics, to study the target and mechanism space of auranofin. This would also allow to assess the power, orthogonality and complementarity of these techniques in the realm of chemical proteomics. TPP is a recently developed technique that can monitor changes in the stability of proteins upon binding to small molecules. Redox proteomics is a method by which the oxidation level of protein cysteinome can be quantitatively analyzed. Auranofin is an FDA-approved anti-inflammatory drug for treatment of rheumatoid arthritis, but due to its potent antitumor activity, it is currently in clinical trials against cancer. Although several MOAs have been suggested for auranofin, uncertainties exist regarding its cellular targets; therefore, this molecule was chosen as a challenging candidate to test the chemical proteomics tools. A combination of the above mentioned tools confirmed thioredoxin reductase 1 (TXNRD1) (ranking 3rd) as the cognate target of auranofin and demonstrated that perturbation of oxidoreductase pathway is the main route of auranofin cytotoxicity. We next showed that changes in the redox state of specific cysteines can be linked to protein stability in TPP. Some of these cysteines were mapped to the active sites of redox-active enzymes. In Paper IV, using quantitative multiplexed proteomics, we helped to show that b-AP15, a bis-benzylidine piperidone compound inhibiting deubiquitinases USP14 and UCHL5, produces a similar perturbation signature as bortezomib in colon cancer cells. However, in comparison with bortezomib, b-AP15 induces chaperone expression to a significantly higher level and leads to a more extensive accumulation of polyubiqutinated proteins. The polyubiqutinated proteins co-localize with mitochondrial membrane and subsequently reduce oxidative phosphorylation. These results help define the atypical cell death induced by b-AP15 and describe why this molecule is effective against apoptosis resistant cells in variety of tumor models. Finally, in Paper V, we extended the applications of TPP and combined it with specificity concept for proteome-wide discovery of specific protein substrates for enzymes. We developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA) that relies on the hypothesis that enzymatic post-translational modification of substrate proteins can potentially change their stability against thermal denaturation. Furthermore, we applied the concept of specificity similar to the above papers, to reveal potential substrates using OPLS-DA. SIESTA was applied to two enzyme systems, namely TXNRD1 and poly-(ADP-ribose) polymerase-10 (PARP10), identifying known and putative candidate substrates. A number of these candidate proteins were validated as PARP10 substrates by targeted mass spectrometry, chemiluminescence and other assays. SIESTA is an unbiased and system wide approach and its broad application can improve our understanding of enzyme function in homeostasis and disease. In turn, specific protein substrates can serve as readouts in high throughput screening and facilitate drug discovery. Taken together, in this thesis, FITExP methodology was improved in two directions. In paper I, we improved the performance of FITExP by combining the proteomics data from detached and attached cells. In Paper II, we demonstrated how the proteomics data on a multitude of drugs in a single cell line enables the discovery of compound targets and MOA. Furthermore, we built an R Shiny package which can serve as a resource for the cancer community in target and MOA deconvolution. In Papers III and IV, we applied an arsenal of chemical proteomics tools for characterization of two anticancer compounds. In Paper V, we expanded the applications of TPP to identification of specific protein substrates for enzymes in a system-wide manner

Training Memory: Exploring the Intersection of Plant Stress Signalling and DNA Methylation

Plants are sessile organisms living in a dynamic environment to which they must continually acclimatize in order to maximise their reproductive potential. This plasticity is achieved through many complex and intricate signalling pathways that allow for the continuous perception, response, and adjustments to new environmental stimuli. A growing body of evidence suggests that such pathways are not merely static but dynamic and can be primed following repeated activation, thus affecting enhanced responses to recurring stresses. Such examples of priming have led to a notion that plants have some capacity to form stress memories of past environmental perturbations. However, the full extent and nature of such memory, and the machinery involved to store and transmit these, remain enigmatic. One prospective mechanism is the involvement of heritable, yet rapid and reversible, chromatin marks that, theoretically, could be shaped by the environment to convey a regulatory effect on the expression of the underlying genotype, thus acting as an epigenetic layer of regulation. This thesis explores the potential intersection of stress signalling pathways and chromatin variation, specifically DNA methylation, to co-ordinate plant stress responses. First, mechanistic insights into the operation of a SAL1-PAP-XRN retrograde signalling pathway to fine-tune plant physiology under drought are presented. A key finding was that this pathway complements canonical ABA signalling to induce stomatal closure, thus minimising water-loss under water limited conditions. Furthermore, the SAL1-PAP-XRN pathway was found to effect chromatin patterns, specifically DNA methylation at short transposable elements. These observations implicate cross-talk with the RNA directed DNA methylation pathway, however, the exact mechanism for this interaction remains to be identified. Multiple investigations were performed to test for stress-induced changes in DNA methylation that could potentially regulate responses to recurring stress, thus conveying a memory. A transgenerational recurring drought stress experiment tested whether descendants of drought-exposed lineages displayed greater drought tolerance (transgenerational memory). For the majority of traits tested, including plant growth rate and drought survival, offspring from plant lineages exposed to successive generations of repeated drought stress performed comparably to those from control lineages. However, memory was demonstrated in the form of enhanced seed dormancy, in drought stressed lineages, that persisted at least one generation removed from stress. Whether this capacity for memory could be related to the type or severity of stress applied, or species examined, remains to be investigated further. The transgenerational drought experiment was paired with a recurring excess-light stress experiment to investigate memory within a generation. Not only did this treatment lead to priming of plant photosynthetic behaviour, indicative of a greater capacity to withstand abrupt increases in light intensity, but new leaves from stressed plants, developed in the absence of stress, also showed altered photosynthetic characteristics compared to unstressed counterparts. Such observations are consistent with the mitotic transmission of stress-induced traits. Given multiple demonstrations of memory, comparisons were made to unstressed controls to test for any correlating changes in DNA methylation that might explain the phenomena observed. However, in both experiments, observations of memory were found to be independent of large-scale conserved changes in DNA methylation discounting it as a conveyor of plant stress memories, under these conditions, raising questions regarding the mechanism(s) responsible for the examples of memory observed herein. Ultimately, this thesis systematically evaluates the notion that plants are able to form genuine memories, potentially underpinned by reversible chromatin marks, that may facilitate acclimation to local environments on a relatively rapid scale compared to the fixation of adaptive genetic polymorphisms. Any capacity for plant stress memories may provide avenues for further epigenomic based agronomic tools to improve crop stress tolerance. However, the nature of such memories observed here appear subtle and nuanced, and are forgotten beyond a generation. Further characterisation and mechanistic understanding of mitotic memory mechanisms, however, may still hold potential. It was also observed that stress signalling pathways can interact with those involved in chromatin modification, giving novel insight into their mechanistic functioning and the how the onset of stress may induce chromatin changes. Despite this potential, the DNA methylome was found to be relatively impervious to stress-induced changes and, thus, is an unlikely memory mechanism