Biology subjects

Bell, T. A.

Publications and source records attributed to Bell, T. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Cross-coronavirus host susceptibility loci influence disease severity through immune mediators

Severe disease following infection with SARS-CoV or SARS-CoV-2 is driven in part by genetically regulated immune responses that promote lung injury. Previously, we showed that genetic risk for severe disease is conserved across viruses and between mouse and human, identifying the HrS43 locus as a shared determinant of severity. Here, to resolve immune pathways linking host loci to disease outcomes, we apply an integrative statistical framework combining Bayesian identification of predictive immune traits with QTL mapping and mediation analysis across infection conditions. This approach identifies immune predictors of disease severity across both viruses, reveals extensive genetic control of the immune system at homeostasis and during infection, and supports locus-specific causal mechanisms of immunopathology. Notably, HrS43 appears to influence disease severity through distinct immune mediators in SARS-CoV versus SARS-CoV-2, demonstrating that conserved genetic susceptibility can drive virus-specific immunopathology with translational relevance across species.

genetics↗

bioRxiv2024

Ancestral sequence reconstruction of Mic60 reveals a residue signature supporting respiration in yeast

In eukaryotes, cellular respiration takes place in the cristae of mitochondria. The mitochondrial inner membrane protein Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS), is crucial for the organization and stabilization of crista junctions and its associated functions. While the C-terminal Mitofilin domain of Mic60 is necessary for cellular respiration, the sequence determinants for this function have remained unclear. Here, we used ancestral sequence reconstruction to generate Mitofilin ancestors up to and including the last opisthokont common ancestor (LOCA). We found that yeast-lineage derived Mitofilin ancestors as far back as the LOCA rescue respiration. By comparing Mitofilin ancestors, we identified four residues sufficient to explain the respiratory difference between yeast- and animal-derived Mitofilin ancestors. Our results provide a foundation for investigating the conservation of Mic60-mediated cristae junction interactions.

evolutionary biology↗

bioRxiv2024

The Updated Mouse Universal Genotyping Array Bioinformatic Pipeline Improves Genetic QC in Laboratory Mice

The MiniMUGA genotyping array is a popular tool for genetic QC of laboratory mice and genotyping of samples from most types of experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve arrays performance and the associated reports usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA in 2020. Here we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for inbred strains and increase the number of constructs that can reliably be detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have made changes to the layout of the report, to simplify the interpretation and completeness of the analysis and added a table summarizing the ideogram. We believe that these changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research.

genetics↗

bioRxiv2023

Cholesterol regulates plasma membrane bending by prominin-family proteins

Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

biochemistry↗

bioRxiv2023

Sarbecovirus disease susceptibility is conserved across viral and host models

Coronaviruses have caused three severe epidemics since the start of the 21st century: SARS, MERS and COVID-19. The severity of the ongoing COVID-19 pandemic and increasing likelihood of future coronavirus outbreaks motivates greater understanding of factors leading to severe coronavirus disease. We screened ten strains from the Collaborative Cross mouse genetic reference panel and identified strains CC006/TauUnc (CC006) and CC044/Unc (CC044) as coronavirus-susceptible and resistant, respectively, as indicated by variable weight loss and lung congestion scores four days post-infection. We generated a genetic mapping population of 755 CC006xCC044 F2 mice and exposed the mice to one of three genetically distinct mouse-adapted coronaviruses: clade 1a SARS-CoV MA15 (n=391), clade 1b SARS-CoV-2 MA10 (n=274), and clade 2 HKU3-CoV MA (n=90). Quantitative trait loci (QTL) mapping in SARS-CoV- and SARS-CoV-2-infected F2 mice identified genetic loci associated with disease severity. Specifically, we identified seven loci associated with variation in outcome following infection with either virus, including one, HrS45, that is present in both groups. Three of these QTL, including HrS45, were also associated with HKU3-CoV MA outcome. HrS45 overlaps with a QTL previously reported by our lab that is associated with SARS-CoV outcome in CC011xCC074 F2 mice and is also syntenic with a human chromosomal region associated with severe COVID-19 outcomes in humans GWAS. The results reported here provide: (a) additional support for the involvement of this locus in SARS-CoV MA15 infection, (b) the first conclusive evidence that this locus is associated with susceptibility across the Sarbecovirus subgenus, and (c) demonstration of the relevance of mouse models in the study of coronavirus disease susceptibility in humans.

genetics↗

bioRxiv2023

Large differences in collateral blood vessel abundance among individuals arise from multiple genetic variants

Collateral blood flow varies greatly among humans for reasons that remain unclear, resulting in significant differences in ischemic tissue damage. A similarly large variation has also been found in mice that is caused by genetic background-dependent differences in the extent of collateral formation, termed collaterogenesis--a unique angiogenic process that occurs during development and determines collateral number and diameter in the adult. Previous studies have identified several quantitative trait loci (QTL) linked to this variation. However, understanding has been hampered by the use of closely related inbred strains that do not model the wide genetic variation present in the "outbred" human population. The Collaborative Cross (CC) multiparent mouse genetic reference panel was developed to address this limitation. Herein we measured the number and average diameter of cerebral collaterals in 60 CC strains, their 8 founder strains, 8 F1 crosses of CC strains selected for abundant versus sparse collaterals, and 2 intercross populations created from the latter. Collateral number evidenced 47-fold variation among the 60 CC strains, with 14% having poor, 25% poor-to-intermediate, 47% intermediate-to-good, and 13% good collateral abundance, that was associated with large differences in post-stroke infarct volume. Genome-wide mapping demonstrated that collateral abundance is a highly polymorphic trait. Subsequent analysis identified: 6 novel QTL circumscribing 28 high-priority candidate genes harboring putative loss-of-function polymorphisms (SNPs) associated with low collateral number; 335 predicted-deleterious SNPs present in their human orthologs; and 32 genes associated with vascular development but lacking protein coding variants. This study provides a comprehensive set of candidate genes for future investigations aimed at identifying signaling proteins within the collaterogenesis pathway whose variants potentially underlie genetic-dependent collateral insufficiency in brain and other tissues.

genetics↗

bioRxiv2022

Forward genetic screen of homeostatic antibody levels in the Collaborative Cross identifies MBD1 as a novel regulator of B cell homeostasis

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL maps to known immunologically important regions of the genomes, Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and show that Methyl-CpG binding domain protein 1 is a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells. SummaryHomeostatic antibody levels are highly variable between Collaborative Cross (CC) mouse strains. This forward genetic screen in the CC identifies Methyl-CpG binding domain protein 1 (MBD1) as a regulator of homeostatic IgG1 levels and marginal zone B cell differentiation.

genetics↗

bioRxiv2022

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.

biochemistry↗

bioRxiv2022

Multi-omics analysis identifies drivers of protein phosphorylation

Phosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated. We quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped [~]700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes.

genomics↗

bioRxiv2022

A Multitrait Locus Regulates Sarbecovirus Pathogenesis

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.

microbiology↗

bioRxiv2022

A natural fusion of flavodiiron, rubredoxin, and NADH:rubredoxin oxidoreductase domains is the highly efficient water-forming oxidase of T. vaginalis

Microaerophilic pathogens such as Giardia lamblia and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain the intracellular redox balance. This oxygen consumption is a result of the H2O-forming NADH oxidase activity of two distinct flavin-containing systems: H2O-forming NADH oxidases (NOXes) and multicomponent flavodiiron proteins (FDPs). Both systems are not membrane-bound and recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment, making them crucial for the survival of human microaerophilic pathogens. In this study, using bioinformatic and biochemical analysis, we show that T. vaginalis lacks a NOX-like enzyme, and instead harbors three proteins that are very close in their amino acid sequence and represent a natural fusion between N-terminal FDP, central rubredoxin and C-terminal NADH:rubredoxin oxidoreductase domains. We demonstrate that this natural fusion protein with fully populated flavin redox centers unlike a "stand-alone" FDP (also present in T. vaginalis), directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of O2 to water within a single polypeptide and with an extremely high turnover. Using single particle electron cryo-microscopy (cryo-EM) we present structural insight into the spatial organization of the FDP core within this multidomain fusion protein. Our studies represent an important addition to our understanding of systems that allow human protozoan parasites to maintain their optimal redox balance and survive transient exposure to oxic conditions.

biochemistry↗

bioRxiv2021

Common Mechanism of SARS-CoV and SARS-CoV-2 Pathogenesis across Species

Sarbecovirus (CoV) infections, including Severe Acute Respiratory CoV (SARS-CoV) and SARS-CoV-2, are considerable human threats. Human GWAS studies have recently identified loci associated with variation in SARS-CoV-2 susceptibility. However, genetically tractable models that reproduce human CoV disease outcomes are needed to mechanistically evaluate genetic determinants of CoV susceptibility. We used the Collaborative Cross (CC) and human GWAS datasets to elucidate host susceptibility loci that regulate CoV infections and to identify host quantitative trait loci that modulate severe CoV and pan-CoV disease outcomes including a major disease regulating loci including CCR9. CCR9 ablation resulted in enhanced titer, weight loss, respiratory dysfunction, mortality, and inflammation, providing mechanistic support in mitigating protection from severe SARS-CoV-2 pathogenesis across species. This study represents a comprehensive analysis of susceptibility loci for an entire genus of human pathogens conducted, identifies a large collection of susceptibility loci and candidate genes that regulate multiple aspects type-specific and cross-CoV pathogenesis, and also validates the paradigm of using the CC platform to identify common cross-species susceptibility loci and genes for newly emerging and pre-epidemic viruses.

microbiology↗

bioRxiv2020

Regulation of protein abundance in genetically diverse mouse populations

Proteins constitute much of the structure and functional machinery of cells, forming signaling networks, metabolic pathways, and large multi-component complexes. Protein abundance is regulated at multiple levels spanning transcription, translation, recycling, and degradation to maintain proper balance and optimal function. To better understand how protein abundances are maintained across varying genetic backgrounds, we analyzed liver proteomes of three genetically diverse mouse populations. We observe strong concordance of genetic and sex effects across populations. Differences between the populations arise from the contributions of additive, dominance, and epistatic components of heritable variation. We find that the influence of genetic variation on proteins that form complexes relates to their co-abundance. We identify effects on protein abundance from mutations that arose and became fixed during breeding and can lead to unique regulatory responses and disease states. Genetically diverse mouse populations provide powerful tools for understanding proteome regulation and its relationship to whole-organism phenotypes. O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/296657v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1c8a941org.highwire.dtl.DTLVardef@e022b6org.highwire.dtl.DTLVardef@106e361org.highwire.dtl.DTLVardef@13a2c2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

AAA+ proteases, which perform regulated protein degradation in all kingdoms of life, consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX must rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP omplexes unfold, translocate, and degrade protein substrates, albeit modestly slower han uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP therefore is ot required for ClpAP protease activity, although some flexibility in how the AAA+ ring ocks on ClpP may be necessary for optimal function.

biochemistry↗

bioRxiv2020

Structural basis of ClpXP recognition and unfolding of ssrA-tagged C-degron substrates

When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In E. coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer-degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

biophysics↗

bioRxiv2020

Content and performance of the MiniMUGA genotyping array, a new tool to improve rigor and reproducibility in mouse research

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well annotated genome, wealth of genetic resources and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost effective, informative and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole genome sequencing. Here we describe the content and performance of a new Mouse Universal Genotyping Array (MUGA). MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: 1) chromosomal sex determination, 2) discrimination between substrains from multiple commercial vendors, 3) diagnostic SNPs for popular laboratory strains, 4) detection of constructs used in genetically engineered mice, and 5) an easy to interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA we genotyped 6,899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived and recombinant inbred lines. Here we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, the extension of the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. There is preliminary but striking evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the main stock for a significant action of the genotyped inbred samples. We conclude that MiniMUGA is a valuable platform for genetic QC and important new tool to the increase rigor and reproducibility of mouse research.

genetics↗

bioRxiv2019

Functionally overlapping variants control TB susceptibility in Collaborative Cross mice

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis (Mtb) infection. We previously found that Collaborative Cross mouse strains differ in their susceptibility to Mtb, and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine, IFN{gamma}, in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 susceptibility. Using a population derived from a CC001/Unc (CC001) x CC042 intercross, we mapped four QTL underlying Tuberculosis ImmunoPhenotypes (Tip1-4). These included 2 major effect QTL on Chromosome 7 (Tip1 and Tip2) that were associated with bacterial burden. Tip2, along with Tip3 (Chromosome 15) and Tip4 (Chromosome 16) also correlated with IFN{gamma} production following infection, whereas Tip1 appeared to control an IFN{gamma}-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and these T cells failed to express the Integrin alpha L (L; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a, and is found within the Tip2 interval. This 15bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 and highlights the multigenic nature of TB control in the Collaborative Cross.\n\nImportance statementThe variable outcome of Mycobacterium tuberculosis infection observed natural populations is difficult to model in genetically homogenous small animal models. The newly-developed Collaborative Cross (CC) represents a reproducible panel of genetically-diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene that is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.

microbiology↗

bioRxiv2019

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

biophysics↗