Browsing by Author "Blignaut, Marguerite"

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    Ataxia telangiectasia mutated protein kinase : a potential master puppeteer of oxidative stress-induced metabolic recycling
    (Hindawi, 2021) Blignaut, Marguerite; Harries, Sarah; Lochner, Amanda; Huisamen, Barbara
    ENGLISH ABSTRACT: Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.
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    Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation
    (Springer Nature, 2019) Blignaut, Marguerite; Loos, Ben; Botchway, Stanley W.; Parker, Anthony W.; Huisamen, Barbara
    The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during β-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.
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    An investigation into the role of ATM protein in mitochondrial defects associated with cardiovascular pathology resulting from insulin resistance
    (Stellenbosch : Stellenbosch University, 2019-04) Blignaut, Marguerite; Huisamen, Barbara; Lochner, Amanda; Engelbrecht, Anna-Mart; Van Vuuren, Derick; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background: Ataxia-telangiectasia (A-T) is a rare, recessive disorder that develops in the absence of Ataxia-Telangiectasia Mutated protein kinase (ATM). This complex disease is characterised by neurodegeneration, increased risk of cancer, a high incidence of insulin resistance and Type 2 diabetes (T2D) as well as cardiovascular disease. Mitochondrial dysfunction is associated with the development of cardiomyopathy and T2D. Obesity and insulin resistance contribute towards the development of cardiac dysfunction, and have been linked with mitochondrial disturbances. ATM has previously been associated with mitochondria, and the absence thereof results in decreased mitochondrial respiration and ATP synthesis as well as structural abnormalities. This study aimed to investigate the role of ATM in mitochondrial oxidative phosphorylation and mitophagy as possible contributors towards cardiovascular dysfunction in obesity. Methodology: The effects of high palmitic and oleic acid, either alone or in combination with insulin or high glucose on ATM expression levels were investigated in an H9c2 cardiomyoblast model. Insulin signal transduction intermediates were determined by western blotting and glucose uptake by [3H]2-deoxyglucose accumulation. Mitoplasts were prepared from cardiac mitochondria of male Wistar rats with digitonin and the quality thereof confirmed with transmission electron microscopy (TEM). The location of ATM was determined with Super-resolution structured illumination microscopy (SR-SIM) and western blotting. Oxidative phosphorylation (oxphos) analysis was performed polarographically (Clark-type electrode) on mitochondria obtained from 1) young male Wistar rat hearts perfused with the ATM specific inhibitor, KU60019 or vehicle (DMSO), and 2) chow fed age-matched controls and diet induced obese (DIO) rat hearts perfused ex vivo with either DMSO or ATM-specific activators and its inhibitor (KU60019 ± insulin or KU60019 ± chloroquine). Oxphos was determined in carbohydrate (glutamate+malate) or fatty acid (palmitoyl-L-carnitine+malate) substrates while protein expression levels of ATM as well as markers of mitophagy and mitochondrial fission were measured (western blotting). The redox status of NAD(P)H was determined with 2-photon fluorescence lifetime imaging microscopy in H9c2 cells ± KU60019. Results: This study showed that: (i) high levels of fatty acids and insulin affects the expression levels of ATM; (ii) ATM is located on the inner mitochondrial membrane of cardiac mitochondria. iii) Inhibition of ATM decreased carbohydrate-stimulated oxidative phosphorylation in cardiac mitochondria (p=0.0024), potentially through Complex I. This was supported by the observation that ATM inhibition decreased NADPH (p=0.02) and increased NADH accumulation in H9c2 cells. iv) The effect of ATM inhibition on oxidative phosphorylation was not influenced by diet; v) ATP synthesis as well as respiratory control index improved with the addition of insulin (p<0.005). vi) The inhibition of ATM was associated with decreased fission (p=0.0038). vii) The decrease in the autophagosomal membrane marker, LC3-II (p<0.0001) seems to be associated with the cytosolic role of ATM. Conclusion: ATM is located on the inner mitochondrial membrane and inhibition thereof influences mitochondrial ATP synthesis, potentially through Complex I substrate oxidation. Inhibition of ATM did not affect oxidative phosphorylation in obesity, but resulted in mitochondrial autophagy disruption as well as decreased fission.
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    The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus A
    (Stellenbosch : University of Stellenbosch, 2009-03) Blignaut, Marguerite; Burger, J. T.; Stephan, D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.
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    Towards a transferable and cost-effective plant AFLP protocol
    (PLoS, 2013) Blignaut, Marguerite; Ellis, Allan G.; Le Roux, Johannes J.
    Amplified fragment length polymorphism (AFLP) is a powerful fingerprinting technique that is widely applied in ecological and population genetic studies. However, its routine use has been limited by high costs associated with the optimization of fluorescently labelled markers, especially for individual study systems. Here we develop a low-cost AFLP protocol that can be easily transferred between distantly related plant taxa. Three fluorescently labelled EcoRI-primers with anchors that target interspecifically conserved genomic regions were used in combination with a single non-labelled primer in our AFLP protocol. The protocol was used to genotype one gymnosperm, two monocot and three eudicot plant genera representing four invasive and four native angiosperm species (Pinus pinaster (Pinaceae), Pennisetum setaceum and Poa annua (Poaceae), Lantana camara (Verbenaceae), Bassia diffusa (Chenopodiaceae), Salvia lanceolata, Salvia africana-lutea, and Salvia africana-caerulea (Lamiaceae)). Highly polymorphic and reproducible genotypic fingerprints (between 37–144 polymorphic loci per species tested) were obtained for all taxa tested. Our single protocol was easily transferred between distantly related taxa. Measures of expected heterozygosity ranged from 0.139 to 0.196 for P. annua and from 0.168 to 0.272 for L. camara which compared well with previously published reports. In addition to ease of transferability of a single AFLP protocol, our protocol reduces costs associated with commercial kits by almost half. The use of highly conserved but abundant anchor sequences reduces the need for laborious screening for usable primers that result in polymorphic fingerprints, and appears to be the main reason for ease of transferability of our protocol between distantly related taxa.