Biology subjects

Brewer, J. R.

Publications and source records attributed to Brewer, J. R..

10 recordsLinked to original sources

bioRxiv2026

A growth-inhibitory dUTPase from Mosigvirus AV110 disrupts cell division and host physiology in Escherichia coli

dUTPases are essential enzymes for nucleotide metabolism as they catalyze the hydrolysis of dUTP to dUMP, thus preventing the toxic incorporation of uracil into DNA. dUTPases are found in all domains of life, including bacteriophages, where they serve multiple functions to support phage propagation. Phage-encoded dUTPases reduce cellular dUTP levels and provide precursors for de novo dTTP synthesis and the synthesis of modified nucleotides, which are important to avoid DNA-based phage defense systems. In this study, we focused on the dimeric dUTPase Gp135, encoded by the Mosigvirus phage AV110, and examined its effect on growth, cell morphology, DNA replication and cell division of AV110s host, extended-spectrum beta-lactamase-producing E. coli. We observed that Gp135 significantly inhibited growth by disrupting DNA replication and cell division. This resulted in cytoplasmic voids, cell elongation, and ultimately cell death. We further demonstrated the diversity of phage-encoded dUTP/dCTPases through phylogenetic analysis, identifying four distinct clusters: one comprising the most common dUTPases, which form trimers; two more divergent clusters comprising dimeric dUTP/dCTPases; and one new cluster of more diverse, uncharacterized dUTPases and dNTPases. Overall, our results highlight the evolutionary and functional diversity of phage-encoded dUTP/dCTPases, with the Mosigvirus AV110 dUTP/dCTPase illustrating their role in supporting phage propagation.

microbiology↗

bioRxiv2026

The Mosigvirus AV110 Elongasome-Modulating Peptide (Emp) inhibits Escherichia coli growth by targeting Mre-dependent peptidoglycan synthesis

Extended-spectrum {beta}-lactamase and AmpC-producing Escherichia coli (ESBL/AmpC E. coli) are increasingly recognized as public health threats due to their resistance to commonly used antibiotics. With limited treatment options available, new antibacterials and antibacterial targets can be explored by studying early-expressed phage proteins involved in host reprogramming, as these proteins interact with and disrupt essential bacterial processes. A systematic screening of 45 early-expressed proteins with unknown function of Mosigvirus AV110 identified six proteins that inhibit the growth of ESBL/AmpC E. coli, suggesting a role in host reprogramming. In-depth analysis of Gp94, termed Elongasome-Modulating Peptide (Emp), demonstrated that it alters cell morphology and the localization of peptidoglycan synthesis. This effect could be partially complemented by MreB and MreD. Since Emp interacts directly with MreB, MreC, and MreD, we propose that Emp perturbs elongasome-associated peptidoglycan synthesis, thus providing a scaffold for developing new antibacterials against ESBL/AmpC-producing E. coli.

microbiology↗

bioRxiv2026

DPP9-mediated inflammasome repression protects against checkpoint inhibitor lung toxicity

Over one million patients receive cancer immunotherapy annually, yet the mechanisms underlying life-threatening immune-mediated toxicities remain poorly understood. Checkpoint inhibitor pneumonitis (CIP) is the leading cause of immunotherapy-related mortality, with a case fatality rate approaching 10%, and no genetic risk factors have been described to date. We identified Dipeptidyl-peptidase 9 (DPP9) as the first genetic susceptibility gene for CIP in a clinico-genomics cohort of 4,397 patients treated with immune checkpoint inhibitors. Mechanistically, DPP9 suppresses CARD8 inflammasome activation and IL-18 secretion in human monocytes, a pathway which is engaged prior to CIP onset, with IL-18 selectively elevated in the plasma of patients who subsequently develop CIP. Myeloid-restricted ablation of Dpp8 and Dpp9 in mice recapitulated the pulmonary histopathological and immunological hallmarks of CIP, including granuloma formation, accumulation of IFN{gamma}-producing T cells and monocyte-derived macrophages. Each of these phenotypes were driven by excessive IL-18 secretion. Together, these findings establish DPP9 as a genetic determinant of CIP and nominate IL-18 blockade as a mechanistically rational therapeutic strategy.

immunology↗

bioRxiv2026

Reverse genetics in humanized mice reveals CARD8-mediated pyroptosis causing pancytopenia in human DPP9 deficiency

Loss of function mutation in the human DPP9 gene causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia and patients require bone marrow transplantation, but the mechanism of cell loss is unclear since Dpp9 mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to little transcriptional changes suggesting post-transcriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to the activation of the CARD8 inflammasome resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

immunology↗

bioRxiv2024

Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups. Here, we compare the potential of derivatives of Nile Red with such functional substitutions for super-resolution fluorescence microscopy of lipid packing in model membranes and living cells. All studied Nile Red derivatives exhibit cholesterol-dependent fluorescence changes in model membranes, as shown by spectrally resolved stimulated emission depletion (STED) microscopy. STED imaging of Nile Red probes in cells reveals lower membrane packing in fibroblasts from healthy subjects compared to those from patients suffering from Niemann Pick type C1 (NPC1) disease, a lysosomal storage disorder with accumulation of cholesterol and sphingolipids in late endosomes and lysosomes. We also find small but consistent changes in the fluorescence lifetime of the Nile Red derivatives in NPC1 cells, suggesting altered hydrogen-bonding capacity in their membranes. All Nile Red derivatives are essentially non-fluorescent in water but increase their brightness in membranes, allowing for their use in MIN-FLUX single molecule tracking experiments. Our study uncovers the potential of Nile Red probes with functional substitutions for nanoscopic membrane imaging.

biophysics↗

bioRxiv2024

IL-4 Licenses B Cell Activation Through Cholesterol Synthesis

Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division1-3. Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands4-6. One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago7. However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo. By limiting lipid availability in vitro, we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.

immunology↗

bioRxiv2023

IL-10 constrains sphingolipid metabolism via fatty acid desaturation to limit inflammation

Unchecked chronic inflammation is the underlying cause of many diseases, ranging from inflammatory bowel disease to obesity and neurodegeneration. Given the deleterious nature of unregulated inflammation, it is not surprising that cells have acquired a diverse arsenal of tactics to limit inflammation. IL-10 is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types; however, the exact mechanism by which IL-10 signaling subdues inflammation remains unclear. Here, we find that IL-10 signaling constrains sphingolipid metabolism. Specifically, we find increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10-deficient macrophages. Genetic deletion of CerS2, the enzyme responsible for VLC ceramide production, limited exacerbated inflammatory gene expression associated with IL-10 deficiency both in vitro and in vivo, indicating that "metabolic correction" is able to reduce inflammation in the absence of IL-10. Surprisingly, accumulation of saturated VLC ceramides was regulated by flux through the de novo mono-unsaturated fatty acid (MUFA) synthesis pathway, where addition of exogenous MUFAs could limit both saturated VLC ceramide production and inflammatory gene expression in the absence of IL-10 signaling. Together, these studies mechanistically define how IL-10 signaling manipulates fatty acid metabolism as part of its molecular anti-inflammatory strategy and could lead to novel and inexpensive approaches to regulate aberrant inflammation.

immunology↗

bioRxiv2022

Nanostructure of the desmosomal plaque

Desmosomes are considered one of the most important intercellular junctions with respect to mechanical strength. Therefore, their spatial distribution and structure is of interest with respect to understanding both healthy and diseased tissue. Previous studies have imaged desmosomes in tissue slices using transmission electron microscopy, or low-resolution confocal images, but both these techniques lack the ability to resolve the 3-dimensional structure of the desmosomes. In this work it was possible to determine the 3D-nanostructure of single desmosomal complexes in both mouse and human epidermis, by 3D stimulated emission depletion (STED) microscopy. STED images of desmoplakin and the desmosomal cadherin, desmoglein revealed that desmosomes form ring-like structures, distributed over the cell surface, with diameters of around 1 m. STED images of the tight junction plaque protein ZO1 also displayed ring formations, suggesting a common structure for intercellular junctions. Measurements of the desmosomal plaque protein, desmoplakin showed an increased intercellular plaque distance during the stratum basale (0.23{+/-}0.027{micro}m) to stratum spinosum (0.28{+/-}0.039 {micro}m) transition.

cell biology↗

bioRxiv2020

The Epidermal Barrier is Indispensable for Systemic Energy Homeostasis

ObjectivesHomeostatic regulation of body temperature is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and neuronal regulators. Although the skin is the largest sensory organ of the human body, and plays a fundamental role in regulating body temperature, it is surprising that adaptive alterations in skin functions and morphology only vaguely have been associated with physiological responses to cold stress or sensation of ambient temperatures. MethodsTo unravel the physiological responses to a compromised epidermal barrier in detail we have used animal models with either defects in skin lipid metabolism (ACBP-/- and skin-specific ACBP-/- knockout mice) or defects in skin structural proteins (ma/ma Flgft/ft). The primary objective was to clarify how defects in epidermal barrier function affect 1) energy expenditure by indirect calorimetry, 2) response to high fat feeding and a high oral glucose load and 3) expression of brown-selective gene programs by quantitative PCR in inguinal WAT (iWAT). ResultsWe show that mice with a compromised epidermal barrier function exhibit increased energy expenditure, increased food intake, browning of the iWAT, and resistance to diet-induced obesity. The metabolic phenotype, including browning of the iWAT, is reversed by housing the mice at thermoneutrality (30{degrees}C) or by pharmacological {beta}-adrenergic blocking. These findings show that a compromised epidermal barrier induces a {beta}-adrenergic response that increases energy expenditure and browning of the white adipose tissue to maintain a normal body temperature. ConclusionOur findings show that the epidermal barrier plays a key role in maintaining systemic metabolic homeostasis. HighlightsEnergy expenditure is significantly augmented in mice with impaired epidermal barrier. Mice with compromised barrier display increased food intake while maintaining normal bodyweight. Mice with an impaired epidermal barrier are resistant to diet-induced obesity and insulin resistance. Compromised barrier function induces expression of brown-selective gene programs in iWAT. Thermoneutral housing or blocking {beta}-adrenergic signaling prevents induction of brite-selective genes in iWAT and reverses food intake.

biochemistry↗

bioRxiv2020

FGF signaling regulates development through combinatorial transduction pathways and by modulating cellular adhesion

FGFs are key developmental regulators which engage a signal transduction cascade through receptor tyrosine kinases, typically involving ERK1/2, PI3K/AKT, and other effectors. However, it remains unknown if all FGF activities depend on kinase activity or these canonical signal transduction cascades. To address these questions, we generated allelic series of knock-in Fgfr1 and Fgfr2 mouse strains, carrying point mutations that disrupt binding of signaling effectors to the receptors, alone or in combination. We also produced a kinase dead allele of Fgfr2 which broadly phenocopies the null mutant. When interrogated in cranial neural crest cells, point mutations in either receptor revealed discrete functions for signaling pathways in specific craniofacial contexts, but failed to recapitulate the single or double null mutant phenotypes even in their most extensive combination. Furthermore, we found that together these signaling mutations abrogated the established FGF-induced signal transduction pathways, yet certain FGF functions such as cell-matrix and cell-cell adhesion remained unaffected. Our studies establish combinatorial roles of both Fgfr1 and Fgfr2 in development and identify novel kinase-dependent cell adhesion properties of FGF receptors, independent of well-established roles in intracellular signaling.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗