Biology subjects

Buchanan, J. L.

Publications and source records attributed to Buchanan, J. L..

3 recordsLinked to original sources

bioRxiv2023

3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle

Tryptophan is an essential amino acid that is extensively characterized as a regulator of cellular function through its metabolism by indoleamine 2,3-deoxygenase (IDO) into the kynurenine pathway. However, despite decades of research on tryptophan metabolism, the metabolic regulatory roles of it and its metabolites are not well understood. To address this, we performed an activity metabolomics screen of tryptophan and most of its known metabolites in cell culture. We discovered that treatment of human colon cancer cells (HCT116) with 3-hydroxykynurenine (3-HK), a metabolite of kynurenine, potently disrupted TCA cycle function. Citrate and aconitate levels were increased, while isocitrate and all downstream TCA metabolites were decreased, suggesting decreased aconitase function. We hypothesized that 3HK or one of its metabolites increased reactive oxygen species (ROS) and inhibited aconitase activity. Accordingly, we observed almost complete depletion of reduced glutathione and a decrease in total glutathione levels. We observed a dose-dependent decrease in cell viability after 48 hours of 3HK treatment. These data suggest that raising the intracellular levels of 3HK could be sufficient to induce ROS-mediated apoptosis. We modulated the intracellular levels of 3HK by combined induction of IDO and knockdown of kynureninase (KYNU) in HCT116 cells. Cell viability decreased significantly after 48 hours of KYNU knockdown compared to controls, which was accompanied by increased ROS production and Annexin V staining revealing apoptosis. Finally, we identify xanthommatin production from 3-HK as a candidate radical-producing, cytotoxic mechanism. Our work indicates that KYNU may be a target for disrupting tryptophan metabolism. Interestingly, many cancers exhibit overexpression of IDO, providing a cancer-specific metabolic vulnerability that could be exploited by KYNU inhibition.

biochemistry↗

bioRxiv2019

Markov modelling of run length and velocity for molecular motors

Molecular motors are nanometer scale proteins involved in various in-tracellular processes such as cargo transport, muscle contraction and cell division. The steps in the chemo-mechanical cycle that use ATP hydrolysis to generate motion along the cytoskeletal tracks can be characterized by stepping rates depending only on the current state, permitting modeling of the cycle as a Markov process. To learn more about the nature of motor mo-tion, cell researchers have conducted in vitro experiments in which molecular motors pull beads along filaments attached to glass slides and their veloc-ity and run length until detachment from the microtubule are recorded. In this article a formula is derived for distance traveled until detachment. Combining this with a standard result for time to absorption for Markov processes gives a formula for velocity. Two kinesins, and two variants of myosin VI, for which there are run length and velocity measurements in the literature are considered. In each case the derived formulas for run length and velocity have a generalized Michaelis-Menten form as functions of ATP concentration. The degrees of the numerator and denominator polynomials increase with the complexity of chemo-mechanical cycle. The degrees of the Michaelis-Menten form determine the maximum number of of reaction rates that can be determined from experimental data on run length and velocity, however consistency conditions may reduce this number and restrict which rates can be determined.

cell biology↗

bioRxiv2018

Energetic stress and infection generate immunity-fecundity tradeoffs in Drosophila

Physiological responses to short-term environmental stress, such as infection, can have long-term consequences for fitness, particularly if the responses are inappropriate or nutrient resources are limited. Genetic variation affecting energy acquisition, storage, and usage can limit cellular energy availability and may influence resource-allocation tradeoffs even when environmental nutrients are plentiful. Here, we utilize well-characterized Drosophila mitochondrial-nuclear genotypes to test whether disrupted energy metabolism interferes with nutrient-sensing pathways, and whether this disruption has consequences for tradeoffs between immunity and fecundity. We find that this energetically compromised genotype is resistant to rapamycin - a drug that stimulates nutrient-sensing pathways that are activated when resources are limited. Resource limitation also compromises survival in energetically-compromised genotypes, suggesting that this genotype may have little excess energetic capacity and fewer cellular nutrients, even when environmental nutrients are not limiting. Accordingly, we find that immune function is compromised in this genotype, but only in females, and that these females experience immunity-fecundity tradeoffs that are not evident in genotypic controls with normal energy metabolism. Thus, genetic variation in energy metabolism may act to limit the resources available for allocation to life-history traits in ways that generate tradeoffs even when environmental resources are not limiting.

genetics↗