Bio21 - Theses
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Deciphering the signalling pathways associated with neuronal death in excitotoxicity by quantitative proteomic approaches
Excitotoxicity is a primary pathological process directing neuronal cell death in both acute neurological disorders and neurodegenerative diseases such as ischemic stroke and Alzheimer’s disease. It is initiated by over-stimulation of ionotropic glutamate receptors (iGluRs), which in turn permit the influx of excessive calcium ions into the cytosol of the affected neurons. The sustained and excessively high cytosolic calcium concentration induces substantial imbalance of the intracellular environment such as depletion of ATP and over-production of reactive oxygen species by inducing dysregulation of specific proteases, protein kinases, and phosphatases. These dysregulated enzymes in turn aberrantly catalyse post-translational modifications of specific neuronal proteins to cause neuronal death. Despite the efforts by many investigators in the past four decades, the cell signalling pathways relaying the neurotoxic signals from the over-stimulated iGluRs and the excessively high cytosolic calcium concentration to direct neuronal death in excitotoxicity, remains unclear. Avenues to chart these neurotoxic signalling pathways include identifying the excitotoxicity-dysregulated proteases, protein kinases, and phosphatases, and delineating the mechanisms of their dysregulation and elucidating their exact roles in excitotoxic neuronal death. In my PhD study, I employed multiple quantitative proteomic approaches and a combination of the neuronal cell-based model and two animal models of excitotoxicity to identify the neuronal proteins aberrantly modified by these excitotoxicity-dysregulated enzymes. Furthermore, these approaches also defined the exact sites of modifications and quantified the degree of modifications. The proteomic approaches I adopted include the quantitative N-terminomic, global and phosphoproteomic approaches. They were used to analyse changes in general protein degradation, limited proteolysis, phosphorylation and dephosphorylation of specific neuronal proteins catalysed by the excitotoxicity dysregulated enzymes in cultured primary cortical neurons and in brain tissues subjected to ischemic stroke and traumatic brain injury. Using an N-terminomics method called “Terminal amine isotopic labelling of substrates” (TAILS), I discovered hundreds of cellular proteins undergoing enhanced proteolytic processing catalysed by the excitotoxicity-activated proteases to form truncated protein fragments in neurons over-stimulated with glutamate. To define the identities of these excitotoxicity-associated proteases, I performed TAILS analysis of neurons co-treated with the excitotoxic level of glutamate and calpeptin, a specific inhibitor of calpains and cathepsins. Results of my analysis revealed that calpeptin abolished enhanced proteolytic processing of the majority of the neuronal proteins identified in the TAILS study of neurons treated with glutamate-only. These findings indicate that most of these identified neuronal proteins were either direct calpain/cathepsin substrates or the substrates of proteases activated by calpains or cathepsins. More importantly, the findings suggest that calpains and cathepsins are the major modulator proteases catalysing proteolytic processing of specific neuronal proteins in excitotoxicity. Using label-free global and phosphoproteomics analyses I found 483 phosphopeptides derived from neuronal proteins undergoing significant changes in phosphorylation level in glutamate-treated primary cortical neurons. Interestingly, global proteomic analysis revealed only a few neuronal proteins exhibiting significant changes in abundance, suggesting that most of the neuronal proteins remained stable up to 240 min of glutamate treatment. These findings suggest that instead of protein synthesis and degradation, phosphorylation plays a major role in modulating the activities and functions of specific neuronal proteins in excitotoxicity. From the identified phosphosites in the neuronal proteins undergoing significant changes in phosphorylation state, bioinformatic analysis predicted PAK1 (Serine/threonine-protein kinase), CK2A1 (Casein kinase II subunit alpha), and mTOR (mammalian target of rapamycin) as the most perturbed kinases in neurons undergoing excitotoxic cell death. Bioinformatic analysis of the neuronal proteins showing significantly changed phosphorylation and/or enhanced proteolytic processing predicted defective axonal guidance signalling as the most perturbed signalling pathway in excitotoxicity. To complement the proteomic analysis in cultured neurons, I conducted phosphoproteomic studies of thebrains in mice suffering from stroke and TBI (traumatic brain injury). Results of my study indicate that neuronal proteins, especially those in synapses are mostly dysregulated. Brain tissue lysates contain proteins expressed in neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells. The high abundance of neuronal proteins identified as the brain proteins exhibiting significant changes in phosphorylation state induced by stroke and TBI suggest that neurons are particularly sensitive to the detrimental impacts of stroke and TBI. The neuronal protein tyrosine kinase Src was discovered by TAILS to be cleaved by calpains to form a truncated Src fragment in excitotoxicity. To examine the therapeutic potential of my proteomic findings, I chose to investigate if blockade of calpain cleavage of Src in neurons could protect against neuronal loss in vivo in a rat model of neurotoxicity. My study showed that stereotaxic injection of a cell membrane-permeable peptide inhibitor of calpain cleavage of Src in rats can protect against neuronal loss induced by over-stimulation of the N-methyl-D-aspartate (NMDA) receptors. Taken together, results of my proteomic analyses have built a conceptual framework for future investigation to decipher the signalling pathways underpinning neuronal death in excitotoxicity. Furthermore, my results provided evidence of the therapeutic potential of the findings from TAILS analysis. Some of the neuronal proteins identified in my study to exhibit significantly perturbed phosphorylation state and/or proteolytic processing, are potential targets for the development of new therapeutic strategies to reduce neuronal loss in ischemic stroke, traumatic brain injury and other neurodegenerative diseases.
The oxidative damage of biological molecules by air pollutants NO2● and NO3●
Air pollution is perceived as the world’s greatest environmental risk to human health. According to the World Health Organization (WHO), air pollution is responsible for the deaths of about 7 million people each year. In the industrialised urban environment, nitrogen dioxide (NO2•) and ground-level ozone (O3) are the most oxidising air pollutants. Exposure to these gases has been associated with increased respiratory health problems, such as exacerbation of existing asthma and allergies. While the adverse health effects of air pollution are clear, the precise underlying mechanism through which the pollutants affect biological systems is not well understood. It has been speculated that nitrate radicals (NO3•), which are formed from the reaction of NO2• and O3, play an important role in the oxidative damage of biological systems. Therefore, this thesis explores reactions involving NO3• and biomolecules such as proteins through a combination of kinetic, computational and product studies, in order to gain a better understanding of the fundamental chemical pathways that lead to oxidative damage in biological systems upon exposure to air pollution. The first section of this thesis investigates the reaction of NO3• with aliphatic amino acids and peptides. From laser flash photolysis experiments, it was found that NO3• reacts with aliphatic amino acids and peptides at multiple sites through proton-coupled electron transfer (PCET) at the amide nitrogen, and hydrogen atom transfer (HAT) at the α-carbon or the activated C–H moiety (e.g., tertiary carbons) with the rate of about 1 x 10(6) M−1 s−1. Following the above finding, this thesis proceeds to examine the reaction of NO3• with aromatic amino acids and peptides. A faster rate by a factor of 5–6 suggests that the reaction occurs at the aromatic ring through electron transfer (ET). An unprecedented amide neighbouring group effect was discovered, by which the rate of aromatic ring oxidation is increased considerably when the ring is flanked by two amide groups, instead of one amide and one ester group. Due to this effect, phenylalanine can potentially act as a relay amino acid in a long-distance ET even though the aromatic ring in phenylalanine is not readily oxidisable under biochemical conditions. The third section of this thesis explores NO3• reactions involving proline, where its side chain is covalently bound to the α-amino group. This unique structure increases electron density at the nitrogen and significantly accelerates the rate of ET at this nitrogen by a factor of about 600 compared to the other aliphatic substrates. However, when the amide moiety in proline residue is involved in the amide neighbouring group effect, accelerating the rate of aromatic ring oxidation, the rate of ET at this nitrogen was found to decrease significantly. The final part of this thesis studies the reaction of NO2• with various biological molecules, including short peptide and cholesterol derivatives. It was found that contrary to the widely accepted radical pathway, the reaction of NO2• with these molecules involves an ionic pathway through the dissociation of N2O4 into NO+ and NO3−.
Investigating the metabolic regulation capacity of the malaria parasite P. falciparum
The pathogenic stages of the malaria parasite Plasmodium falciparum develop within red blood cells, where they have access to an abundant supply of glucose. Unsurprisingly, these parasite stages are heavily reliant upon glucose for meeting their energy and biomass requirements, which is catabolized in the glycolytic and pentose phosphate pathways. While these pathways also exist in host cells, there is increasing evidence that P. falciparum has evolved novel ways for regulating glucose metabolism that could be targeted by the next generation of antimalarial drugs. During my PhD, I studied one such level of metabolic regulation, which occurs via metabolic repair enzymes regulation of central carbon metabolism, in blood stages of P. falciparum. The glyoxalase system is ubiquitous across organisms and facilitates the detoxification of methylglyoxal – generated via incorrect functioning of the glycolytic enzyme triose-phosphate isomerase – into D-lactate. I found the cytosolic glyoxalase enzymes to be responsible for the majority of detoxification of methylglyoxal, as disruption of the cytosolic glyoxalase I (GloI) impairs D-lactate production. In addition, I discovered a novel end-product to this pathway, the unusual metabolite phospho-D-lactate. In addition, I characterised a member of the haloacid dehalogenase family of metabolite phosphatases, the phospho-glycolate phosphatase (PGP), which regulates phospho-D-lactate levels. Parasite mutants lacking the PGP enzyme are viable but exhibit diminished growth rates in red blood cells. They accumulate phospho-D-lactate, as well as 4-phosphoerythronate – a putative side product of another glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase – suggesting PGP acts upon these two metabolites at minima. Metabolically, the accumulation of phospho-D-lactate and 4-phosphoerythronate was associated with changes in flux through the glycolytic and pentose phosphate pathways. Moreover, parasites with a non-functional PGP enzyme exhibited an increased sensitivity to the antimalarial fosmidomycin. Our findings suggest that metabolic end/side products of glycolysis directly regulate the metabolism of P. falciparum, and that the intracellular levels of these metabolites are tightly controlled by metabolic repair enzymes.
Investigation of the mechanisms of action of and resistance to artemisinin and other endoperoxide antimalarials
P. falciparum malaria caused 219 million cases with an estimation of 435,000 deaths in 2017. Artemisinin (ART) combination therapies (ACTs) reduce the parasite load swiftly and have contributed to decreasing malaria deaths. However, the emergence and spread of ART- resistance in South-East Asia is threatening global health. ART-resistance manifests as a reduced susceptibility to ART at the early-ring stage in parasites carrying mutations in the PfK13 protein. However, the details of the molecular mechanism of resistance remain unclear. Several lines of evidence indicate that ART resistance is associated with altered protein homeostasis. However, there is no method available to quantitate protein turnover in P. falciparum. First, I established pulse-SILAC (stable isotope labelling with amino acid in cell culture) (pSILAC), a proteomics-based approach, to quantify the rates of protein turnover across the P. falciparum proteome. I applied pSILAC to provide the first detailed analysis of protein turnover across the intraerythrocytic developmental cycle (IDC). The data showed that protein turnover increases as the parasites develop, peaking at the trophozoite stage and decreasing at schizont stage. At the individual protein level, proteins belonging to different groups demonstrate different turnover profiles across the IDC. To investigate how PfK13 mutations help parasites withstand ART-induced toxicity, I applied pSILAC to examine protein turnover in a PfK13 mutant strain (Cam3.IIR539T) and a wildtype revertant (Cam3.II_rev) strain across the ring stage of development before and after DHA exposure. The data showed that protein turnover is similar in the Cam3.IIR539T and Cam3.II_rev lines in the absence of DHA. Following exposure of early ring-stage parasites (0-8 h.p.i.) to a clinically relevant DHA (3-h, 700 nM), both strains underwent disruption of protein turnover, which was further supported by an increased level of eIF2α phosphorylation (an ER stress marker). Interestingly, Cam3.IIR539T re-initiated protein turnover at the late ring stage. This increase in protein metabolism represents the earliest (so far) detected cellular event that distinguishes PfK13 mutant and wildtype parasites and was independently validated using an (unstable) firefly luciferase-based reporter system that enables monitoring protein turnover and re-synthesis. A quantitative proteomic approach showed that the level of expression of PfK13 is ~2-fold less in the Cam3.IIR539T strain than the Cam3.II_rev strain at both ring and trophozoite stages. To examine whether this decreased level of PfK13 underpins the altered sensitivity of ring-stage parasites to DHA, I made use of a transfectant in which 90% PfK13 can be inducibly mis- localized from the cytoplasm to the nucleus (90% KS). Following a 3-h, 700 nM DHA pulse, ~20% of the 90% KS parasites survived, compared with ~0% of the wildtype strain. The data suggest that decreased abundance of PfK13 directly underpins decreased sensitivity to DHA, even in the absence of any mutations. Lastly, I examined the efficacy of two endoperoxide antimalarials with longer in vivo half-lives, OZ277 and OZ439, against PfK13 mutant parasites. I showed that OZ277 and OZ439 are activated in vivo via the same mechanism as DHA (i.e. by haem derived from haemoglobin digestion). When parasites were exposed to the different endoperoxides for periods that are relevant to the in vivo half-lives of the drugs, OZ277 and OZ439 are much more effective against PfK13 mutant parasites than DHA.
Structural analysis of critical interactions in the replication machinery of rabies virus
Rabies virus is a non-segmented negative sense RNA virus that causes encephalitis in humans with a 100% case fatality rate, resulting in > 61,000 deaths/year world-wide. There are currently no treatments for rabies disease, but a number of critical interactions of viral proteins provide potential targets to develop new antiviral compounds. Of particular interest are the interactions of viral nucleo (N), phospho (P), and large/polymerase (L) wherein the N protein encapsidates genomic RNA to form the helical nucleocapsid (N-RNA) that serves as the template for viral transcription and replication by the RNA-dependent polymerase complex, composed of the enzymatic L protein and non-catalytic polymerase cofactor P protein. P protein is critical in attaching L to the N-RNA template via an interaction between the P protein C-terminal domain (P-CTD) and the C-terminal trypsin-sensitive peptide of N protein (N-pep), which provides a potentially valuable target for anti-viral drug design. As a multifunctional protein, P also forms multiple interactions with host factors that underlie diverse roles in the virus-host interface, particular in immune evasion. Its C-terminal domain (P-CTD) alone bears binding sites for host factors including STAT1, microtubule and PML protein and two phosphorylation sites, apart from N-binding site. The strategies by which P mediates diverse functions in viral replication and immune evasion by coordinating its multiple interactions with viral protein and host cellular proteins remain unknown. We have commenced a project to investigate the structure and dynamics of P-CTD and characterize its precise molecular interactions with N-pep. The P-CTD and N-pep have been expressed in Escherichia coli, generating protein at high yield and purity. NMR titrations of P-CTD with N-pep suggest a single binding mode with a micromolar affinity at the positive patch of P-CTD and the flexible loop region of N-pep. A mutagenesis study indicates that the interaction between N-pep and P-CTD is mainly mediated through electrostatic and hydrophobic interactions. By introducing mutations and cyclizing N-pep, the binding affinity has been improved 200-fold. The structural and dynamic study of P-CTD and its phosphomimetics using X-ray crystallography and NMR suggests that there is no conformational change of P-CTD upon phosphorylation. NMR titration experiments further confirmed that the single binding site model of P-CTD/N-pep is not affected by the phosphorylation state of P-CTD.
Spatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor
Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.
The impact of proteostasis imbalance on proteome solubility
A hallmark of neurodegenerative diseases is that certain proteins abnormally aggregate into insoluble deposits. A leading hypothesis is that a breakdown in protein folding quality control mechanisms leads to the accumulation of unfolded or misfolded proteins that are prone to aggregation. The hypothesis of the thesis is that there would be a metastable sub-proteome vulnerable to aggregation when protein quality control is stressed by any mechanism. However, a systematic understanding of the proteins that are affected remains poorly understood. Here extensive quantitative proteomic studies were performed to measure the changes in proteome abundance and proteome solubility (as determined by 100,000 g pelleting for 20 min) arising from different triggers of proteostasis stress that have reported roles in leading to protein misfolding and aggregation. These included three specific inhibitors of key proteostasis hubs (Hsp70, Hsp90 and the proteasome), two exogenous stresses (oxidative stress and ER stress) related to neurodegenerative diseases and aggregation of mutant Huntingtin exon1 protein in a mouse neuroblastoma cell model of Huntington’s disease. Unexpectedly, all stresses led to no detectable change in net proportion of aggregated protein, suggesting that the proteome is robustly buffered against the accumulation of misfolded protein. However, at the individual protein level, there were many changes both upwards and downwards for all stresses. Most changes can be ascribed to a functional remodelling of protein complexes involved in adaptation to stress rather than protein misfolding with stress granule proteins being centrally involved. The comparison of proteome solubility changes across all stresses revealed that different proteostasis stresses yielded highly distinct solubility signatures. Although no common metastable sub-proteome was detected, proteins that were sensitive to stress shared similar physicochemical features. More insoluble proteins were enriched with high molecular weight, low isoelectric point and prion-like domains. By contrast, more soluble proteins were enriched with low molecular weight and low-complexity domains. Indeed, assessment of proteome-wide solubility changes offers richer power into mechanisms than the standard measurement of protein abundance levels. Therefore, it is suggested this methodology can be used more generally alongside standard quantitative proteomic analyses of protein levels to gain deeper insight to molecular function of diverse biological mechanisms.
It’s hip to be square: a cyclobutene diester approach to alkyl citrate natural products
This thesis features an enatiospecfic synthesis of a key alkyl citrate retron that was leveraged in the total syntheses of squalene synthase inhibitors (-)-CJ-13,982, (-)-CJ-13,981 and (-)-L-731,120 (featured in Org. Let. 2018, 20, 4255–4258). This key retron was prepared in 7 linear steps, requiring only 4 purification, with a 40% yield from (S)-(+)-γ-hydroxymethy-γ-butyrolactone. The synthesis highlights the application of a formal [2+2] cycloaddition and a remarkable acid-mediated rearrangement sequence to furnish the correct stereochemistry and oxidation level of the citrate moiety. This thesis demonstrates the shortest enantiospecifc total synthesis of (-)-CJ-13,981 to date, via the use of this key citrate retron, affording this natural product in 7.7% total yield over 10 steps. Efforts towards the squalene synthase inhibitor (-)-L-731,120 and the viridiofungins, a family of serine palmitoyl transferase inhibitors that have activity inhibiting hepatitis C replication, are also featured.
In vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster
Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
Characterisation of the human TIM22 mitochondrial import translocase
Mitochondria are essential cellular organelles for cell viability due to their fundamental role in ATP production, programmed cell death and biosynthetic pathways. Dysfunctional mitochondria are implicated in various pathologies including cancers, cardiovascular diseases and neurodegenerative diseases. Mitochondrial function relies on ~1500 mitochondrial proteins, which are predominately nuclear-encoded and must be imported into mitochondria. Protein trafficking to mitochondria is executed by sophisticated multimeric protein import machines, termed, Translocases. One such machine, the Translocase of the Inner Membrane 22 (TIM22) mediates the import of an important class of hydrophobic proteins, the carrier proteins, which facilitate chemical exchange across the inner membrane, contributing to cellular metabolism and bioenergetics. While TIM22 has been well-characterised using Baker’s yeast, very little is known about the human complex. This study has focussed on disentangling the molecular composition of the human TIM22 complex, and mechanisms underpinning the carrier import pathway. Using immunoprecipitation/mass-spectrometric analysis, we uncovered two novel, metazoan-specific subunits of the human TIM22 complex, termed Tim29 and Acylglycerol kinase (AGK). This is the first report of additional subunits of the human TIM22 complex since the initial reports of the human complex in 1999 (Bauer et al., 1999b). We showed that Tim29 is involved in the assembly of the TIM22 complex and creates contacts with the general entry gate of the outer membrane, the TOM complex, providing a novel mechanism for the translocation and import of hydrophobic proteins. Dissecting the molecular function of AGK revealed a lipid kinase-independent role of this previously described mitochondrial lipid kinase in mediating carrier protein import via the TIM22 complex. Identification of AGK at TIM22 also shed additional insight into the pathomechanisms underpinning Sengers syndrome, a mitochondrial disorder uniquely associated with AGK mutations, providing an unexpected link between mitochondrial protein import and Sengers syndrome. The TIM22 complex is also linked to a neurodegenerative disease, Mohr-Tranebjaerg syndrome (MTS), which is caused by mutations in the TIMM8A gene. To date, the pathomechanism underlying this disease remain unknown. Using CRISPR/Cas9-genome editing and label-free quantitative proteomics, we analysed two distinct cell lines (HEK293 and SH-SY5Y) and revealed a cell-specific function of hTim8a in Complex IV biogenesis in neuronal mitochondria. Our findings indicate that loss of hTim8a leads to mitochondrial dysfunctions which amplifies cytochrome c levels in mitochondria and sensitises cells to death. This research has contributed new insights into understanding of the human TIM22 translocase and carrier protein biogenesis. It has revealed novel features and knowledge on the pathomechanism of two TIM22-associated mitochondrial diseases – Sengers syndrome and Mohr-Tranebjaerg syndrome.
Fitness trade-offs involved in adaptation to host plants, diapause-egg phenotypes and pesticides pressure in the redlegged earth mite, Halotydeus destructor
The redlegged earth mite, Halotydeus destructor, is an invasive species introduced into Australia from South Africa. In Australia, this species enters summer diapause at the egg stage which can survive desiccation, heat exposure and applications of pesticides over summer, while annually it produces approximately three generations of active mites in the cool and wet period. Hatching of diapause eggs approximately synchronize the germination of annual plants and the post-diapause mites (1st generation), thus causing serious damage on seedlings. Understanding the mechanism and fitness cost of adaptation to host plants, environmental changes and pesticides pressure in this species is crucial for development of an integrated pest management (IPM) strategy. My experiment reveals that host performance of H. destructor is influenced by the interaction between mite populations and host plants. Mite survival, net reproductive output, development and feeding damage on plants are reduced if the previous host plant of a population is different from the host plant of the introduced microcosm, revealing that a fitness cost is involved in host adaptation. A typical type of diapause eggs with a thick chorion is known to survive sprays of pesticides and stressful summer. A program, Timerite®, has been designed in Western Australia to predict the onset of the typical diapause (TD) egg production based largely on daylength, and farmers spray pesticides according to Timerite® predicted date to suppress populations of pre-diapause mites. However, effectiveness of Timerite® is less in eastern areas of Australia than Western Australia. My study reveals a cryptic type of diapause (CD) egg which lacks a thick chorion and thus is morphologically similar to the non-diapause (ND) egg. Production of diapause eggs (including both TD and CD types) was triggered by longer-daylength, as well as hotter and drier conditions in comparison with conditions leading to ND eggs. However, CD egg can be produced under shorter daylength and cooler temperatures in comparison with TD eggs. Coexistence of CD and ND eggs reflects a bet-hedging strategy in that some individuals can enter diapause for unpredictable conditions. Nevertheless, a fitness penalty of this strategy is revealed by lower diapause intensity of CD eggs when parental mites were reared in cooler and moister conditions. Heavy reliance of synthetic pyrethroid chemicals targeting active mite has led to target-site resistance (kdr) evolving. The L1024F substitution in the voltage-gated para sodium channel is incompletely recessive. Heterozygote (RS) individuals which are detectable in genetic screening are therefore undetectable by bioassays. Only the resistant homozygote (RR) survive 100 mgL-1 bifenthrin which is the discriminating dose and also the field rate, while RS heterozygotes do not survive. Refuges untreated by pyrethroids could reduce resistance evolution because susceptible homozygous (SS) mites in refuges mate with RR survivals in sprayed areas to produce RS. Furthermore, the resistant population has decreased net reproductive output in comparison with susceptible populations, revealing a fitness cost of the L1024F substitution. The frequency of resistant alleles in a population is therefore decreased in the absence of pyrethroid chemicals.
Synthesis and kinetics of novel ionic liquid soluble hydrogen atom transfer reagents
The use of radical methodologies has been greatly developed in the last 50 years, and in an effort to continue this progress, the reactivity of radical reactions in greener alternative solvents is desired. The work herein describes the synthesis of novel hydrogen atom transfer reagents for use in radical chemistry, along with a comparison of rate constants and Arrhenius parameters. Two tertiary thiol-based hydrogen atom transfer reagents, 3-(6-mercapto-6-methylheptyl)-1,2-dimethyl-3H-imidazolium tetrafluoroborate and 2-methyl-7-(2-methylimidazol-1-yl)heptane-2-thiol, have been synthesised. These are modelled on traditional thiol reagents, with a six-carbon chain with an imidazole ring on one end and tertiary thiol on the other. 3-(6-mercapto-6-methylheptyl)-1,2-dimethyl-3H-imidazolium tetrafluoroborate comprises of a charged imidazolium ring, while 2-methyl-7-(2-methylimidazol-1-yl)heptane-2-thiol has an uncharged imidazole ring in order to probe the impact of salt formation on radical kinetics. The key step in the synthesis was addition of thioacetic acid across an alkene to generate a tertiary thioester, before deprotection with either LiAlH4 or aqueous NH3. Arrhenius plots were generated to give information on rate constants for H-atom transfer to a primary alkyl radical, the 5-hexenyl radical, in ethylmethylimidazolium bis(trifluoromethane)sulfonimide. A comparison of the results from the Arrhenius studies for both charged and uncharged t-thiols reveal no significant difference between rate constants (1.16 × 107 M-1 s-1 vs. 1.11 × 107 M-1 s-1 respectively), pre-exponential factors or activation energies. When comparing to the commonly used t-BuSH, the rate constant at 25 °C for the uncharged 2 methyl-7-(2-methylimidazol-1-yl)heptane-2-thiol is essentially identical within experimental error, while the rate constant at 25 °C for the charged 3-(6-mercapto-6-methylheptyl)-1,2-dimethyl-3H-imidazolium tetrafluoroborate is marginally faster than for t-BuSH under the same conditions. An IL-supported organostannane 1-(6-diphenylstannyl-hexyl)-2,3-dimethyl-3H-imidazolium tetrafluoroborate was also synthesised, modelled on the commonly used triphenylstannane reagent. Literature precedence exists for similar tin hydride compounds, however, the synthetic route involved steps with reproducibility issues. The synthesis was dramatically improved by utilising a hydrostannylation reaction, with other steps optimised to allow easier synthesis and purification. An Arrhenius plot was also generated for 1-(6-diphenylstannyl-hexyl)-2,3-dimethyl-3H-imidazolium tetrafluoroborate in ethylmethylimidazolium bis(trifluoromethane)sulfonimide and compared to the traditional tributyl and triphenylstannane Arrhenius parameters. The rate constant for hydrogen atom transfer from this novel stannane to a primary alkyl radical at 25 °C was found to be 5.01 × 106 M-1 s-1. Similar to the thiol salt, this result is marginally faster than the equivalent result for the common reagent, tributylstannane.