Towards Executable Biology

Heringa, J. [Promotor]Fokkink, W.J. [Promotor]Feenstra, K.A. [Copromotor

Foundation technologies in synthetic biology: tools for use in understanding plant immunity

The plant hormone salicylic acid (SA) is an essential activator of plant immune responses directed against biotrophic pathogens. The transcription cofactor NPR1 (Nonexpressor of pathogenesis- related (PR) genes 1) functions to transduce the SA signal into an operational response directed to limited pathogen damage. In the absence of pathogen, NPR1 protein resides in the cytoplasm as a large molecular weight oligomer held together by disulphide bonding. Initiation of defence signalling leads to changes in intracellular redox conditions that promote NPR1 momomer release. Translocation of monomeric NPR1 to the nucleus results in the activation of over 2200 immune-related genes in Arabidopsis. NPR1 lacks a canonical DNA-binding domain but is known to perform part of its regulatory function through engagement of TGA factors (bZIP transcription factor). Induction of SA-dependent signalling is invariably associated with PR-1 gene expression and accumulation of mRNA for this gene serves as a useful marker of defence activation. However, both functional redundancy and stochastic factors limit the effectiveness of standard genetic approaches used in plant research, and thus much of the hierarchal processes surrounding NPR1-dependent gene activation are not fully understood. Using a synthetic biology approach we aim to complete exploratory work and set the foundations for the development of a yeast tool that can be used to manipulate and subsequently understand NPR1 function in relation to interacting partners and gene activation. Accordingly, using this tool we sought to create a conceptual protein circuit based on theoretical plant immunity. In completing this work we have developed a Saccharomyces cerevisiae strain that exhibits a highly oxidising intracellular redox environment. This was achieved by knocking out genes encoding S-nitrosoglutathione reductase (SFA1), flavohemoglobin (YHB1) and YAP1 (bZIP transcription factor), all important components in regulating cellular redox homeostasis and protein S-nitrosylation state in S. cerevisiae. Characterisation of this cell (designated Δsfa1yap1yhb1) reveals a high tolerance to such redox perturbations. Importantly, NPR1 is by default, assembled predominantly in the oligomeric form in this biological chassis. By activating two inducible inputs in the form of Arabidopsis S-nitrosoglutathione reductase (AtGSNOR) and Thioredoxin (AtTRXh5) which both function to promote NPR1 monomerisation, we have created a switch to selectively control NPR1 oligomer-monomer equilibrium. To complete the synthetic circuit, TGA3 was included, along with a modified yeast MEL1 promoter that has been customised to contain the TGA-responsive upstream activation sequence (termed the as-1 element) present in the promoter region of the PR-1 gene. Using FRET tools we were able to confirm nuclear interaction between monomeric NPR1 and TGA3, with this association appearing to induce as-1 element binding. However this process is not sufficient to activate a Luciferase (LUC) reporter gene, even when the GAL4 activation domain (GAL4 AD) is fused to NPR1. Ordinarily, a CUL3-dependent proteolysis-coupled transcription cycle is necessary to maintain efficient NPR1-dependent gene transcription in Arabidopsis. Although S. cerevisiae encodes an evolutionarily related CUL3 ortholog, examination by western blot demonstrates that NPR1 protein is stable in this cell, indicating an endogenous mechanism to degrade NPR1 is either not present or not functional in yeast. As such, this synthetic yeast tool represents a completely novel approach to identify missing components functioning in NPR1-mediated transcriptional regulation. Furthermore, in collaboration with a skilled bioinformatician, and using a rule-based stochastic modeling tool known as Kappa, we have been able to develop, for the first time, a preliminary mathematical simulation representative of NPR1-dependent gene activation that can be used as a foundation for future works

Molecular Cloning and Functional Characterization of Brassica UBC13 Genes

Lysine63 (K)-linked polyubiquitination of target proteins is a fundamentally different process from conventional K48-linked polyubiquitination that targets proteins for degradation via the 26S proteosome. Lys63-linked polyubiquitination regulates numerous cellular processes. The unique feature of Ubc13 compared to other ubiquitin-conjugating enzymes (Ubcs) is its ability to form a stable complex with a Ubc-E2 variant (Uev), which promotes the formation of Lys63-linked polyubiquitination. Ubc13 functions in DNA damage tolerance in budding yeast and is involved in several pathways in mammalian cells. Arabidopsis contains two UBC13 genes and four UEV1 genes that are involved in various developmental processes and stress responses including DNA damage response, root development and immunity. Recent studies imply that AtUbc13s contribute to plant susceptibility against soil-borne pathogen such as clubroot, a major disease in Brassica napus. However, there is no published information regarding characterization of B. napus Ubc13s (BnUbc13s). This project aims to understand functions of Ubc13 and Ubc13-Uev1 complexes in canola. As canola is a polyploid and often contains many homologous genes, this study aims to provide guidelines to selectively target a subset of homologous genes by gene editing to protect from clubroot disease. Twelve BnUBC13 genes were identified through genomic data analysis, eight of which encode proteins different from AtUbc13s were cloned and characterized. All eight BnUbc13s were able to physically interact with AtUev1 to form stable complexes. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects, suggesting that BnUBC13s can replace yeast UBC13 in DNA damage tolerance. Furthermore, a CRIPSR/Cas9 construct was designed to simultaneously target five BnUBC13 genes and was used to transform B. napus cv. Westar (DH12075). Twenty-eight out of thirty regenerated lines were found to contain homozygous or heterozygous mutations in 5 targeted BnUBC13 genes, validating our genomic editing approach in canola. In addition, BnUBC13 transcript levels in resistant and susceptible canola before and after clubroot infection were analyzed based on the in-house RNA-seq data and were found to not fluctuate drastically. This study provides convincing data to support notions that B. napus Ubc13s promotes Ly63-linked polyubiquitination, that BnUbc13s are involved in error-free DNA damage tolerance and that BnUBC13s are housekeeping genes

Structural and Signaling Elements Important for the Efficient Degradation of BHMT through Macroautophagy

Healthy cells maintain a dynamic and responsive intracellular environment that is marked by the synthesis and degradation of proteins, complex macromolecules and organelles. Autophagy, literally self-eating, is a mechanism that delivers cellular cargo to the lytic compartment for digestion. Defects in the regulation of autophagy have been implicated in pathologies such as cancer and neurodegenerative disease, making the study of its regulation compelling. However, few studies have looked at the regulation of mammalian autophagy as a function of a specific cargo protein. Previous studies had indicated that the metabolic enzyme betaine homocysteine methyltransferase (BHMT) is degraded through an autophagic mechanism. One aim of this study has centered on the role of BHMT quaternary structure in determining the efficiency of autophagic sequestration and degradation. In these studies, an oligomerization deficient form of BHMT was used to show that modulation of the Class III PI3 kinase signaling pathway is likely involved in discerning monomeric from multimeric BHMT and that this has a role in the subsequent degradation of BHMT by autophagy. The second aim has been to study to role of the nutrient-regulated mTOR pathway in the autophagic degradation of BHMT. It has been proposed that mTOR-mediated inactivation of S6 kinase is required for induction of autophagy in mammals. However in Drosophila melanogaster, S6 kinase activity has been shown to be essential for induction of autophagy. The current study demonstrates that the inhibitory signal from mTOR to autophagy does not go through S6 kinase or subsequent phosphorylation of the ribosomal protein S6. The significance of these observations in terms of misfolded proteins, neurodegenerative diseases and therapeutics is discussed

PROTEIN FOLDING AND DISEASE THE MITOCHONDRIAL PROTEIN FRATAXIN

This dissertation focuses on the study of frataxin, a small mitochondrial protein whose deficiency is associated with the neurodegenerative disease Friedreich's ataxia (FRDA). Aiming at a better understanding of frataxin conformational and functional properties, two lines of research were followed: first, the effect of FRDA-related mutations in human frataxin (FXN) were studied and the role of oxidative stress related modification addressed; second, yeast frataxin (Yfh1) orthologue was used to explore the conformational and functional properties of the protein.(...)Fundação para a Ciência e a Tecnologi

The role of phosphatidylinositol 3-kinases in autophagy regulation

Autophagy requires the biogenesis of autophagosomes (APs), which are large multilamellar vesicles that sequester cytoplasmic substrates and undergo a maturation process that ultimately leads to their fusion with lysosomes. Previous studies have suggested that local production of phosphatidylinositol-3-phosphate (PI3P) by class III phosphatidylinositol 3-kinase (PI3K) (i.e., Vps34) is required for AP biogenesis at specialized sites of the endoplasmic reticulum called "omegasomes". Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. To address this question, we generated Vps34 knock-out mouse embryonic fibroblasts (MEFs) and analyzed the impact of Vps34 deletion on autophagy in mammalian cells. Using a novel higher affinity 4x-FYVE finger PI3P-binding probe, we found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in the Vps34 knock-out MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. In addition, we used a lipidomic approach to capture the lipid profile of cells in the presence and absence of Vps34 under steady-state and during starvation-induced autophagy. Lipidomics is an emerging powerful tool with the potential to identify new interconnected metabolic lipid networks as well as generate new hypotheses. Here, we identified a new relationship between Vps34 and cholesterol homeostasis. Additionally, we identified specific changes in lysolipids during autophagy. Lastly, we investigated whether the retromer complex plays a role in autophagy. Retromer is a protein complex that binds PI3P on the endosomal membrane and mediates retrograde trafficking of transmembrane proteins from the endosome to the trans-Golgi network. Recent studies have shown a downregulation of this complex associated with sporadic Alzheimer's disease (AD) and have demonstrated aberrant trafficking and processing of APP, a pathological feature of AD, as a result of retromer deficiency. Because retromer is important for maintaining endo-lysosomal system function, we hypothesized that it promote efficient autophagy and may contribute to the dysfunctional autophagy observed in AD when impaired. Using standard autophagy assays, such as assessing LC3 conjugation and puncta formation, our preliminary studies suggest a negative regulatory role for retromer in autophagy. Additionally, we observed a strong association of retromer with Atg9, an autophagy-related gene transmembrane protein that is believe to traffic lipids to the growing autophagosome membrane and recycle autophagy proteins from this compartment

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Analysis of the nuclear proteome of the resurrection plant Xerophyta viscosa (Baker) and its response to dehydration stress

Xerophyta viscosa Baker (family Velloziaceae) can survive extremes of dehydration (desiccation), down to 5% relative water content (RWC) and resumes full physiological activity within 80 h of rehydration. A thorough understanding of this phenomenon may provide further insight into possible mechanisms for improving drought tolerance in other plants. In this respect a comprehensive analysis of the nuclear proteome of this plant and its response to dehydration stress at 35% RWC was carried out. The RWC at 35% represents a distinct phase of the dehydration process where induction of late protection mechanisms is initiated and is a characteristic of desiccation tolerant species. We optimized nuclei isolation and nuclear protein extraction protocols and successfully employed these protocols to isolate highly purified nuclei and subsequently nuclear proteins from fully hydrated and dehydrated X. viscosa leaf samples. The integrity of the purified nuclei was confirmed with light and fluorescent microscopy. The nuclei were uniform spheres, approximately 5 μm in size. The purity and enrichment of the nuclear proteins were confirmed by chlorophyll assay and Western blot analysis. The nuclear proteins were investigated using two-dimensional (2D) and isobaric tags for relative and absolute quantitation (iTRAQ) technologies. Using the 2DE approach, a total of 438 proteins spots were reproducibly detected and analysed of which 18 protein spots were shown to be up-regulated in response to dehydration. These proteins contained both regulatory and functional proteins. The largest category comprised five novel protein factors and two proteins with unassigned functions. The second category comprised proteins involved in gene regulation and signal transduction. The third category comprised stress responsive proteins with chaperone type activities. Other categories include proteins involved in energy metabolism, protein degradation and translation. These results demonstrate that dehydration was controlled by multiple genes within the plant nucleus and X. viscosa may possess its own specific nuclear proteins that are involved in desiccation stress. In addition we comprehensively analyzed the nuclear proteome of X. viscosa using iTRAQ with two-dimensional liquid chromatography and tandem mass spectrometry to complement the data obtained from the 2DE approach. Using iTRAQ, we reproducibly University of Cape Town identified 128 proteins with confidence ¥ 95% (Ï < 0.05). Sixty six percent of the identified proteins showed consistent expression levels. The remaining 34% proteins showed significant changes in expressions. Of the latter, 23% were shown to be up regulated in response to dehydration stress. The remaining 11% were shown to be down regulated. The nuclear proteins of X. viscosa up-regulated in response to dehydration stress showed a coordinated response involving both regulatory and functional proteins and were implicated in diverse cellular functions. The characteristic feature of the X. viscosa nuclear proteins is the high level of stress molecules among the dehydration responsive proteins with evident functions in defense mechanisms compared to down regulated proteins and proteins showing consistent expression levels. These results demonstrate that enhanced defense capacity is crucial to desiccation tolerance and strongly support the notion that late dehydration responsive proteins are involved in protection of the cellular structures during dehydration. Proteins showing consistent expression levels during dehydration most likely maintain the minimum viability in cells under all conditions or may be indirectly associated with desiccation tolerance. Down-regulated proteins are likely important for plant survival under normal growth conditions. The proteins up-regulated in response to dehydration stress were assumed to be associated directly with the acquisition of desiccation tolerance. The up-regulated proteins were further categorized into nine functional groups to gain more insight into their roles in desiccation tolerance. The largest group was shown to be involved in gene regulation and signal transduction (36%), which reflects the role of the nucleus in gene expression and regulation. The second group included stress responsive molecules such as antioxidants, molecular chaperones and compatible solutes (33%). This reflects the importance of strong defense systems in preventing lipid peroxidation, protein aggregation, membrane leakage and maintaining the integrity of cellular structures during dehydration and in the dried state. The third group contained proteins involved in nucleocytoplasmic transport (10%). This might reflect the capacity of this plant to control the movement of molecules to and from the nucleus during dehydration and the importance of this process in adaptation to dehydration stress. The fourth group contained proteins involved in protein translation (7%). Proteins categorized to other functions, include proteins with miscellaneous and unknown functions. Proteins with unknown functions were considered to be X. viscosa nuclear-specific proteins. There was good correlation between the up-regulated proteins identified by 2-DE and iTRAQ approaches. In conclusion, this study revealed that X. viscosa nuclear proteome was responsive to dehydration stress and desiccation tolerance is University of Cape Town genetically encoded. Secondly, X. viscosa relies on readily inducible protection to combat desiccation and desiccation tolerance is controlled by multiple genes within the plant nucleus. Thirdly, the protective mechanisms of desiccation tolerance utilized by X. viscosa appear to involve signal perception genes and modulating gene expression of appropriate genes encoding protective molecules including antioxidants, molecular chaperones, compatible solutes, proteins of translation and degradation machinery, proteins with miscellaneous functions and novel protein factors. Lastly, proteins are crucial to desiccation tolerance allowing X. viscosa to possess a unique stress tolerance with versatile and coordinated actions to provide protection for its cellular structures during desiccation and in the dried state. To our best knowledge this is the first study to provide insight into the nuclear (organellar) proteome of a desiccation tolerant plant