Biology subjects

Walsh, D. J.

Publications and source records attributed to Walsh, D. J..

4 recordsLinked to original sources

bioRxiv2024

IspE Kinase as an Anti-infective Target: Role of a Hydrophobic Pocket in Inhibitor Binding

Enzymes of the methylerythritol phosphate (MEP) pathway are potential targets for antimicrobial drug discovery. Here we focus on 4-diphosphocytidyl-2-C-methyl-D-erythritol (IspE) kinase from the MEP pathway. We use biochemical and structural biology methods to investigate homologs from the pathogenic microorganisms; Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii. We determined the X-ray structures of IspE-inhibitor complexes and studied selected inhibitors binding modes targeting the substrate pocket. The experimental results indicate the need for distinct inhibitor strategies due to structural differences among IspE homologs, particularly for A. baumannii IspE, which displays unique inhibitory profile due to a tighter hydrophobic subpocket in the substrate binding site. This study enhances our understanding of the MEP enzymes and sets the stage for structure-based drug design of selective inhibitors to combat pathogenic microorganisms.

biophysics↗

bioRxiv2023

Convergent generation of atypical prions in knock-in mouse models of genetic prion disease

Most cases of human prion disease arise due to spontaneous misfolding of wild-type or mutant prion protein. Though recapitulating spontaneous prion conversion in animal models has proven challenging, transgenic mice expressing the misfolding-prone bank vole prion protein (BVPrP) recreate certain key aspects of sporadic and genetic prion disease. However, it remains unclear whether spontaneous prion generation can occur in the absence of protein over-expression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knock-in mice expressing physiological levels of either wild-type or mutant BVPrP with isoleucine at codon 109. While mice expressing wild-type BVPrP remained free from neurological disease, a subset of knock-in mice expressing BVPrP with mutations that cause either fatal familial insomnia (D178N) or familial Creutzfeldt-Jakob disease (E200K) developed progressive neurological illness. Brains from spontaneously ill knock-in mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant prion protein. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP knock-in mice transmitted disease to mice expressing wild-type BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical both pre- and post-transmission, suggesting that both mutations guide the formation of a highly similar atypical prion strain. These findings imply that knock-in mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. Therefore, these mice represent useful tools for studying the early stages of genetic prion diseases.

neuroscience↗

bioRxiv2023

Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice as they age.

One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genomes impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false positive results. Here we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial related disease and the influence of nutrients on DNA mutation rates. Significance StatementUsing a sensitive method for mitochondrial heteroplasmy analysis, we show that both vitamin B12 and folic acid can impact mitochondrial DNA mutation. This effect requires further investigation of the potential impact on humans.

molecular biology↗

bioRxiv2022

Mito-SiPE: A sequence-independent, PCR-free mitochondrial DNA enrichment method for ultra-deep sequencing that minimises amplification and alignment artifacts.

BackgroundDeep sequencing is often used to measure somatic variation in the mitochondrial genome. Selective enrichment methods, such as PCR amplification or probe hybridization/capture are commonly used. These methods can introduce bias and are prone to contamination by nuclear-mitochondrial sequences (NUMTs); elements that can introduce artefacts into analyses such as an assessment of mitochondrial heteroplasmy. ResultsHere, we demonstrate a method to obtain ultra-deep (>80,000X) sequencing coverage of the mitochondrial genome by selectively purifying the intact organelle itself using differential centrifugation and alkaline lysis. We applied this approach to seven different mouse tissues. Isolation of mitochondria yields a preparation of highly enriched mtDNA. We compared this method to the commonly used PCR-based method. Mito-SiPE avoids false-heteroplasmy calls that occur when long-range PCR amplification is used for mtDNA enrichment. DiscussionWe have described a modified version of a long-established protocol for purifying mtDNA and have quantified the increased level of mitochondrial DNA post-enrichment in 7 different mouse tissues. This method will enable researchers to identify changes in low-frequency heteroplasmy without introducing PCR biases or NUMT contamination that are falsely identified as heteroplasmy when long-range PCR is used.

genomics↗