1,721,005 research outputs found
Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand
No full textWe have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (Kd 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites
Kinetic investigations of the oxygen entry pathway of the hypoxia-inducible factor (HIF) prolyl hydroxylase 2
Get PDFHuman hypoxia inducible factor (HIF) is responsible for mediating the body’s response to low O2 availability. The levels and activity of HIF are regulated by four Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases, prolyl-hydroxylase domains 1-3 (PHD 1-3) and factor-inhibiting HIF (FIH), collectively termed the HIF hydroxylases. The PHDs catalyse hydroxylation of specific prolyl residues in the N - and C - terminal oxygen-dependent degradation domains of HIF-α, targeting it for degradation by the proteasome. FIH catalyses hydroxylation of an asparaginyl residue in the C-terminal transactivation domain, preventing the interaction of HIF-α with the co-transcriptional activator p300. Under hypoxic conditions, the activity of the oxygen-dependent HIF hydroxylases is reduced, causing an increase in cellular HIF-α levels/activity and triggering the transcription of genes that enable the cellular and physiological hypoxic response. PHD2 is reported to be the key O2 sensor regulating the hypoxic response. It has also been reported to have a high Km(O2) value and a slow reaction with O2 in pre-steady state studies, kinetic features that are proposed to be related to its role as an O2-sensor. We are interested in the molecular features that enable this O2-sensing role, when other Fe(II)/2OG oxygenases react ∼100-fold more rapidly with O2. To investigate whether restricted O2 passage to the active site may be responsible for its unusual kinetics, our collaborators (C. Jorgensen and C. Domene, KCL) have conducted molecular dynamic studies to investigate the route of O2 entry to the PHD2 active site. These studies (unpublished) have indicated that O2 enters PHD2 via the interface between HIF substrate and a flexible, substrate-binding loop (the β2β3 loop) in PHD2. O2 is then proposed to reside in a stable 'E-cluster' in PHD2, before moving to the active site. This report describes work conducted to experimentally verify this predicted O2 entry pathway into PHD2, and to determine whether aspects of this entry pathway contribute to the slow kinetics of the reaction of PHD2 with O2. To validate the role of the β2β3 loop in the reaction of PHD2 with O2, kinetic studies of PHD2 loop variants were undertaken. These variants were previously developed by Flashman et al such that the β2β3 loop of PHD2 was altered to include the loop sequences of PHD1 and PHD3. These loop variants demonstrated reduced O2 sensitivity, and with the β2β3 loop of PHD3, a more rapid reaction with O2. These data suggest the β2β3 loop of PHD2 does indeed play an important role in O2 uptake. Experimental validation of the stable E-cluster was then undertaken. The site directed mutagenesis of methionine 299 in PHD2 to histidine did not yield different kinetics with respect to O2, suggesting either replacement of methionine 299 with histidine does not alter the O2 'stabilising' characteristics of the E-cluster or the E-cluster is not significant in O2 kinetics. Tryptophans 258 and 389 also form part of the E cluster. Laser-induced excitation of molecular O2 to O2* was used to attempt to modify and thus identify amino acid residues in regions where O2 was stably bound, i.e. the E-cluster. This approach was technically challenging, and results obtained were inconclusive. Further investigations using this method would require substantial optimisation. Stopped-flow tryptophan fluorescence quenching studies confirmed that these residues encounter a quenching species upon introduction of O2, supporting the hypothesis that they are present on the O2 uptake pathway. Interestingly, the rate of tryptophan fluorescence quenching of the PHD2variant.CODD.Fe(II).2OG complex by O2 correlates with the PHD2 WT rate of product turnover suggesting the quenching is correlated with product turnover. The results reported here imply that the rate limiting step with respect to PHD2 and O2 is prior to O2 reaching the E cluster. Overall the thesis supports the hypothesis that PHD2's β2β3 loop is involved in PHD2's O2–sensing capability. Experiments were unable to verify the existence of an E-cluster, including the finding that O2 encounters two tryptophans present along the proposed O2 uptake pathway in PHD2 at the same rate as PHD2 catalysis. The findings of this study suggest that the β2β3 loop of PHD2 may be a key molecular feature involved in O2 sensing by PHD2, a factor that could be taken into account when designing PHD2 inhibiting or activating therapies
Modulation of Arabidopsis thaliana plant cysteine oxidase activity
No full textAerobic organisms use O2 sensing systems to tailor cellular activities to O2 availability. Low O2 (hypoxic) environments are generated in plant tissues at different stages of development and during submergence stress. In higher plants, O2 sensing machinery includes a family of non-heme Fe- and O2 dependent thiol dioxygenase enzymes, the Plant Cysteine Oxidases (PCOs) and Group VII Ethylene Response Factor (ERF-VII) transcription factors that promote the expression of hypoxia-responsive genes. PCOs modulate the stability of ERF-VIIs based on O2-availability. Under normal O2 conditions (normoxia), PCOs catalyse the oxidation of the N-terminal cysteine of ERF-VIIs. The resultant Cys-sulfinic acid is an N-degron and proceeds along the Arg N-degron pathway for degradation. Conversely, hypoxia limits PCO activity due to the lack of O2 thereby stabilising ERF-VIIs. ERF-VIIs translocate to the nucleus and their transcriptional activities shift the plants’ metabolism to an anaerobic mechanism.
Temporary ERF-VII stabilisation has been associated with improved submergence tolerance. Flooding events are becoming more frequent as a result of climate change. There is evidence to suggest that artificial stabilisation of ERF-VIIs prior to submergence primes plants, thereby improving their tolerance to the subsequent hypoxic stress.1 Chemical inhibition of PCOs has been proposed as a means to achieve this. This thesis identifies peptide and small molecule modulators of PCO activity, explores their mechanism of action and determines outcomes of their application in planta.
In vitro assays monitoring turnover of a truncated Cys-initiating ERF-VII substrate, RAP2.122-15/17 by recombinant Arabidopsis thaliana PCO4 (AtPCO4) allowed for screening of peptides and small molecule modulators. In Chapter 2, a series of ERF-VII based peptidomimetics with modified N-termini were screened and were found to increase AtPCO4’s catalytic activity towards RAP2.122-15/17. Following confirmation that this did not arise from non-specific peptide AtPCO4 interactions, mode of activation investigations also discounted peptidomimetic induced O2 preparation at the active site. Building evidence highlighted the potential involvement of a disordered loop region that is suspected to play a role in substrate binding. Whilst PCO activation is not expected to offer direct agrochemical benefit, the structure-activity insights gleaned could identify a mechanism by which PCO activity could be targeted and reduced.
In Chapter 3, two libraries of small molecules were screened for AtPCO inhibition in in vitro assays and yeast- and plant-based bio assays. Nine candidates from the first library showed in vitro potency that did not translate in vivo. 5/1199 candidates from the second library showed success in vivo by stabilising a RAP2.122-28 linked luciferase reporter in Arabidopsis thaliana seedlings, inducing upregulation of hypoxia-responsive genes under normoxia and improving survival of seedlings following an anoxia treatment. IC50 determination in vitro confirmed the specificity of 2 of these candidates for AtPCO4. This work fulfilled the objectives of this thesis by identifying the first known inhibitors of PCOs and offering lead candidates in the development of an agrochemical for improving plant flood tolerance
Engineering a plant cysteine oxidase as a strategy to improve flood tolerance of plants
No full textPlant cysteine oxidases (PCOs) serve as O2 sensors in plants by catalysing dioxygenation of the N-terminal cysteine (Nt-Cys) of their substrates. The substrates are subsequently degraded via the N-degron pathway. The largest substrate group of PCOs consists of Group VII Ethylene Response Factors (ERFVIIs). Under low oxygen concentrations (hypoxia), e.g., during floods, the activity of PCOs is suppressed, facilitating the stabilisation of ERFVIIs which in turn induce the expression of hypoxia adaptation genes. Thus, the O2 sensitivity of PCOs directly affects the hypoxia and flood response of plants. The aim of the work described in this thesis was to understand and manipulate the substrate scope and the O2 sensing mechanism of AtPCO4, the most O2 sensitive paralogue in Arabidopsis thaliana. Modifying AtPCO4 could extend the stabilisation of its substrates during hypoxia and thereby may increase the flood tolerance of plants. A structure-guided approach was used to design active site and O2 tunnel mutants of residues potentially relevant for AtPCO4 activity. First, the protein construct for recombinant protein synthesis was optimised (Chapter 2). Then, active site mutants were designed by comparing the crystal structures of AtPCO4 with its human homologue ADO, due to its different O2 sensitivity, and a cysteine dioxygenase, due to its different substrate scope. AtPCO4 variants were synthesised with mutation of residues potentially important for iron-coordination (H98D, H100D), interactions with the Nt-Cys substrate (V105G/I, S107L, D177E), catalysis mechanism (O2 activation / transfer, intermediate stabilisation; I175F, C173A) or coordination of the protein substrate at the likely substrate entrance (Y73R, Y183F, Figure 1). The expression levels of the variants and the wildtype enzyme were similar and all recombinant enzymes were stable in solution. The variants were characterised in vitro (Chapter 3), and the results confirmed that the iron-coordinating His-triad is essential for AtPCO4 activity. Residues potentially close to the Nt-Cys substrate, V105, D177 and S107, influenced the catalytic mechanism by affecting iron / substrate positioning, while the residues further back in the active site, C173 and I175, appeared to impact the catalysis due to effects on O2 activation / transfer and / or intermediate stabilisation. Residues at the substrate entrance, Y73 and Y183, proved to be essential for substrate binding. Variants C173A and Y183F were further investigated in an Arabidopsis plant model (Chapter 4) due to their reduced activity caused by different effects on catalysis (Figure 1). In planta, the reduced AtPCO4 activity of both variants could increase the transcription of hypoxia adaptation genes in normoxia. This could be reversed by a higher transcription of AtPCO4. Submergence experiments indicated that lower AtPCO4 activity may improve Arabidopsis tolerance especially when the hypoxia response is not elevated in normoxia. A possible O2 tunnel in AtPCO4 was investigated biochemically by synthesising variants with a potentially widened tunnel entrance (Chapter 5). The results suggested that increasing the tunnel entrance height elevated the O2 entry rate, while increasing the tunnel entrance width might elevate the O2 exit rate. The next step would be to examine the effects of these variants in the Arabidopsis model. Overall, the results in this thesis support the hypothesis that engineering the catalytic properties of AtPCO4 in vitro can lead to improved flood tolerance of plants. Future work is required to validate the submergence results and to investigate if engineering the O2 tunnel of AtPCO4 could tailor the submergence tolerance further. The most promising mutations could then be translated into crop PCOs for improved submergence tolerance
DIRECTED EVOLUTION AND FUNCTIONAL EVALUATION OF PLANT CYSTEINE OXIDASE VARIANTS IN YEAST AND PLANTS
No full textIn normoxic conditions, PCO (Plant Cysteine Oxidase) enzymes catalyse the oxidation of ERF-VII (Ethylene Response Factors) transcription factors. This is an oxygen-dependent step of the N-degron pathway that will lead to the degradation of the ERF-VIIs. In hypoxia, low levels of oxygen lower PCO activity, ERF-VIIs are stabilized and can initiate the expression of genes involved in the hypoxic response.
The engineering of PCOs has gained increasing interest due to the greater extremes of stress, such as submergence, that plants have to face as a result of climate change. Our aim is identify PCO variants with reduced activity, leading to faster stabilization of ERF-VIIs resulting in a better response to submergence.
To address this, different strategies were used. We first followed a rational design, and tested different PCO4 variants that could hinder its activity due to structural variations either in the oxygen tunnel or its active site. We also set-up preliminary experiments to exploit directed evolution based on random mutagenesis, using yeast as a host.
We tested two pre-existing variants, already shown biochemically to reduce PCO activity, in Arabidopsis thaliana by submerging them for 2.5 and 3.5 days and comparing their survival and recovery rate with those of the wild-types. The two variants showed higher survival rates, consistent with previous experiments and biochemical evaluations.
For functional assessment, we implemented a method for the high-throughput screening of PCO variants activity based on luminescence in yeast. The activity of each variant was measured by comparing the relative Fluc activity after 6 hours of (aerobic or hypoxic) treatment with that of the wild type and a negative control. 12 PCO variants of interest were tested with this in vivo platform.
This yeast assay is a useful tool for the rapid, cost-effective, large-scale screening of PCO variants in an in vivo platform and in a controlled environment before moving to more complex plant models.
Finally, we developed a construct for the selection in yeast of interesting PCO variants using a previously characterized system based on zeocin resistance and 5FdU susceptibility. This system can be used to find novel interesting PCO variants with a high-throughput selection from a pool of randomly mutagenized PCOs, solely based on their activity. This approach can also facilitate the discovery of new residues of interest for manipulating PCO activity.
These approaches provide valuable tools to further investigate the functionality of PCOs through both random and rational method
Plant cysteine oxidase function in early plants and algae
Get PDFAll aerobic organisms require O2 for survival. When O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. While plants can survive temporary periods of acute hypoxia, prolonged exposure can result in plant damage or death. A concern over food security has driven higher plant hypoxia research, with little contributed to understanding marine or early land plants. The photosynthesis of phytoplankton is the dominant global process that replenishes atmospheric and oceanic O2. In light of global warming, and the reported expansion of oceanic O2 minimums, the impact oceanic deoxygenation will have on algae, or vice versa, must be assessed.
The higher plant hypoxia response is mediated by the group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs). ERF-VII transcription factor stability is regulated by the Arginine/Cysteine (Cys/Arg) branch of the N-degron pathway. It has been shown that the O2-dependent Plant Cysteine Oxidases (PCOs) catalyse oxidation of ERF-VII N-terminal cysteine residues to form cysteine-sulfinic acid. This renders the ERF-VII N-terminus a substrate for arginyl-tRNA protein transferase (ATE); arginylation promotes ubiquitination by E3 Ubiquitin ligase PRT6 (PRT6) and degradation by the proteasome. During hypoxia, the ERF-VII transcription factors are stabilised, resulting in upregulation of genes which promote survival. Genes encoding for putative PCOs are ubiquitous across the plant kingdom. However, the ERF-VII transcription factors are confined to the flowering plants.
Undoubtedly, there are metabolic challenges associated with living as single-celled aquatic organisms, and also with transitioning from single-celled phytoplankton to land-dwelling, multicellular plants. What role the PCOs might have played in this transition, or in what capacity the PCOs help regulate algal health with respect to O2 has yet to be described. This thesis uses a combination of recombinant protein studies, PCO substrate discovery and algal culturing to investigate the evolution of the plant O2 sensing pathway, the effect of hypoxia on algal health, and the role of algal PCOs. To address these questions, a series of model plant species were selected for study. M. polymorpha is a liverwort, with liverworts reputed to be the first land plants; possibly retaining features of its algal ancestors and extant land plants. K. nitens is an undifferentiated freshwater alga and is a model for understanding the plant transition from water to land. E. huxleyi is a globally distributed Haptophyte, with extraordinary adaptive capabilities and therefore a powerful tool for understanding algal adaptation to climate change-driven stresses. All plants contain putative PCOs, with K. nitens and M. polymorpha also containing ATE and PRT6 homologs – essential elements of the Cys/Arg branch of the N-degron pathway.
This thesis describes novel PCO enzymes from Marchantia polymorpha (MpPCO) and Klebsormidium nitens (KnPCO). MpPCO was characterised with an endogenous substrate, MpERF, with homology to the ERF-VII transcription factors. Despite being the only PCO isoform in M. polymorpha, MpPCO catalytic activity was relatively insensitive to changes to O2 concentrations. By contrast, KnPCO had the greatest O2 sensitivity of any PCO characterised thus far. KnPCO did not have as broad a substrate profile as other PCOs, failing to oxidise LITTLE ZIPPER 2, a known substrate of MpPCO and the higher plant PCOs. This thesis also established algal hypoxia culturing facilities and identified potential hypoxia relevant proteins in the C. reinhardtii proteome, which were verified as substrates of the PCOs in vitro.
This study takes the first steps towards understanding the relationship between the basal and higher plant O2 sensing pathways. It characterises a functional PCO with an ERF-VII-related substrate in M. polymorpha, and describes the first algal PCO and substrates. It suggests an O2 sensitive N-degron pathway exists in K. nitens, without any ERF-VII, ZPR2 or MC-initiating VRN2 substrates in the K. nitens genome. Substrate hunting experiments suggest an algal metabolic response to hypoxia, which could have far reaching consequences for global algal health
Kinetic and mechanistic studies of oxygen sensing Fe(II)/2-oxoglutarate dependent oxygenases
Get PDFThe Fe(II)/2-oxoglutarate (2OG) dependent oxygenases are a widespread enzyme family, which are characterised by structurally similar active sites and proposed to employ a common reaction mechanism. The work described in this thesis concerned kinetic and biophysical studies on 2OG oxygenases, with a particular focus on the hypoxia-inducible transcription factor (HIF) hydroxylases and mechanistic aspects of their reaction with oxygen. The four human HIF hydroxylases regulate cellular levels and transcriptional activity of HIF by catalysing its post-translational hydroxylation in response to changes in oxygen availability. The three prolyl hydroxylase domain enzymes (PHDs1-3) and factor inhibiting HIF (FIH) are proposed to act as cellular oxygen sensors and provide a direct link between oxygen availability and the hypoxic response. Previous transient kinetic studies have shown that PHD2 (the most important human PHD isoform) reacts slowly with oxygen, a factor proposed to be related to its oxygen-sensing role. The molecular mechanisms for the slow PHD2 reaction with oxygen were investigated using a range of kinetic and biophysical techniques to probe the effects of key active site substitutions. The studies reveal that a conservative substitution to an Fe(II)/H2O binding residue results in 5-fold faster reaction with oxygen, suggesting a role for H2O release from the active site in limiting the ability of oxygen to react with PHD2. This thesis also describes the first transient kinetic studies of FIH. The obtained results show that the rate of the FIH reaction with oxygen was significantly faster than for PHD2. Further, FIH catalyses hydroxylation not only of HIF-α, but also of proteins containing ankyrin repeat domains (ARD). The rate of the FIH reaction with oxygen was shown to be substrate dependent; faster oxygen activation of the reaction in the presence of ARD compared with HIF substrates was observed. Mechanistic studies were performed to investigate a report that PHD2 is involved in the enzymatic oxidation of an oncometabolite (R)-2-hydroxyglutarate (2HG) to give 2OG, in what would be an unprecedented reaction for a 2OG oxygenase. This work found that 2HG does not substitute for 2OG in PHD2 catalysis. Instead, the non-enzymatic transformation of 2HG to 2OG was observed, which could potentially contribute to the reported 2HG-dependent PHD activation in vivo. The biophysical and transient kinetic techniques used for studying the HIF hydroxylases were also applied to study the mechanism of deacetoxycephalosporin C synthase (DAOCS, the enzyme catalysing penicillin N ring expansion). Previously, it has been suggested that the DAOCS mechanism differs from the consensus 2OG oxygenase mechanism. The results described in this thesis provide strong evidence that DAOCS employs the consensus ordered mechanism characteristic of 2OG oxygenases, supporting the proposal that the consensus mechanism is a common feature of the 2OG oxygenase family. Overall, the work described in this thesis is supportive of the proposal that most, if not all, 2OG oxygenases employ a common mechanism. However, the differences in the kinetics of their reaction with oxygen, presented throughout the thesis, suggest that different 2OG oxygenases have different rate-limiting steps. Thus, the kinetics of specific oxygenases may be adapted to their biological function, in particular that of PHD2 as the key cellular O2 sensor
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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