Biology subjects

Bundy, J. G.

Publications and source records attributed to Bundy, J. G..

5 recordsLinked to original sources

bioRxiv2024

Plasma membrane transporter Progressive Ankylosis Protein Homolog (ANKH/Ankh) mediates senescence-derived extracellular citrate and is regulated by DNA damage, inflammation and ageing

A considerable body of recent evidence supports citrate transport as a major regulator of organismal lifespan and healthspan. Citrate accumulates outside senescent cells in vitro and in vivo. However, the detailed mechanism of senescent cell extracellular citrate (EC) accumulation is not clear. We show here that EC is partially mediated by a newly described plasma membrane citrate transporter ANKH/SLC62A1 (progressive human ankylosis - ANKH) in senescent fibroblasts. Analogous to interleukin 6 (IL-6), EC and/or ANKH are regulated by telomere dysfunction, the p38 mitogen-activated kinase axis, transforming growth factor beta and p53, but in contrast not by steroids, sodium butyrate, or Ataxia Telangiectasia Mutated (ATM). ANKH was upregulated in other senescent cell types relevant to ageing but not keratinocytes. In contrast, EC and ANKH were inhibited in dividing and senescent fibroblasts by interleukin 1 (IL-1) in parallel with increased IL-6 secretion. Loss- and gain of function mutations of ANKH/Ank are associated with disease and interestingly, Ank is also downregulated in both aged mouse liver and brain tissues in parallel with increased senescence markers and several cytokines, suggesting that inflammatory cytokines could inhibit EC production in vivo. These data identify ANKH/Ank as a novel regulator of senescence-derived EC in both humans and mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/609895v3_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1eed642org.highwire.dtl.DTLVardef@1349040org.highwire.dtl.DTLVardef@1ea867aorg.highwire.dtl.DTLVardef@22d251_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Telomerase deficiency in humans is associated with systemic age-related changes in energy metabolism

Underlying mechanisms of plasma metabolite signatures of human ageing and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis Congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, DKC1) and progressive telomere attrition. We show extracellular citrate is repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo; leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, citrate and senescence factors only weakly distinguished DC patients from controls, whereas other tricarboxylic acid cycle metabolites, lactate and especially pyruvate distinguished them with high significance, consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data has similarities with age-related disease signatures in humans. Our results have implications for the early diagnosis of telomere dysfunction and anti-senescence therapeutics. HighlightsO_LIExtracellular citrate is regulated by telomere function in vitro and in vivo. C_LIO_LIDyskeratosis Congenita (DC) is a human disease characterized by systemic telomere attrition, which showed an age-related plasma energetic profile, distinct from age-related disease and that of centenarians. C_LIO_LIThe DC profile strikingly out-performed senescence factors in discriminating DC from controls, and pyruvate associated with a low lactate:pyruvate ratio is potentially a useful and cheap minimally invasive diagnostic aid for DC and telomere dysfunction. C_LIO_LIMechanistically DC systemic metabolism is indicative of a shift to reduced pyruvate dehydrogenase activity, glycolysis and/or increased citrate and lactate production followed by further metabolism in the kidneys and liver. C_LI

cell biology↗

bioRxiv2021

Pathway analysis in metabolomics: pitfalls and best practice for the use of over-representation analysis

Over-representation analysis (ORA) is one of the commonest pathway analysis approaches used for the functional interpretation of metabolomics datasets. Despite the widespread use of ORA in metabolomics, the community lacks guidelines detailing its best-practice use. Many factors have a pronounced impact on the results, but to date their effects have received little systematic attention in the field. We developed in-silico simulations using five publicly available datasets and illustrated that changes in parameters, such as the background set, differential metabolite selection methods, and pathway database choice, could all lead to profoundly different ORA results. The use of a non-assay-specific background set, for example, resulted in large numbers of false-positive pathways. Pathway database choice, evaluated using three of the most popular metabolic pathway databases: KEGG, Reactome, and BioCyc, led to vastly different results in both the number and function of significantly enriched pathways. Metabolomics data specific factors, such as reliability of compound identification and assay chemical bias also impacted ORA results. Simulated metabolite misidentification rates as low as 4% resulted in both gain of false-positive pathways and loss of truly significant pathways across all datasets. Our results have several practical implications for ORA users, as well as those using alternative pathway analysis methods. We offer a set of recommendations for the use of ORA in metabolomics, alongside a set of minimal reporting guidelines, as a first step towards the standardisation of pathway analysis in metabolomics. Author summaryMetabolomics is a rapidly growing field of study involving the profiling of small molecules within an organism. It allows researchers to understand the effects of biological status (such as health or disease) on cellular biochemistry, and has wide-ranging applications, from biomarker discovery and personalised medicine in healthcare to crop protection and food security in agriculture. Pathway analysis helps to understand which biological pathways, representing collections of molecules performing a particular function, are involved in response to a disease phenotype, or drug treatment, for example. Over-representation analysis (ORA) is perhaps the most common pathway analysis method used in the metabolomics community. However, ORA can give drastically different results depending on the input data and parameters used. In this work, we have established the effects of these factors on ORA results using computational simulations applied to five real-world datasets. Based on our results, we offer the research community a set of best-practice recommendations applicable not only to ORA but also to other pathway analysis methods to help ensure the reliability and reproducibility of results.

bioinformatics↗

bioRxiv2020

Calm on the surface, dynamic on the inside. Molecular homeostasis in response to regulatory and metabolic perturbation of Anabaena sp. 7120 nitrogen metabolism

Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. {Delta}amt mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. The analytical data highlights several internal adaptations, including increased N assimilation (i.e. greater GS activity) and nitrogenase activity (i.e. faster activation upon N deprivation) together with altered amino acids metabolism, as indicated by changes in Gln, Glu and 2-OG, indicating an altered C/N balance. The analyses provides evidence for an active role of AMT transporters in the regulatory/signalling network of N metabolism in this biological system, and the existence of a novel fourth IF7A-independent regulatory mechanism controlling GS activity.

systems biology↗

bioRxiv2017

GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation

Ammonium assimilation in E. coli is regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase (GS), the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolved in vivo concentrations of metabolites, total and post-translationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of GS, GlnB and GlnK under time-varying external ammonium level in the wild type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.

systems biology↗