Biology subjects

Karpowicz, P.

Publications and source records attributed to Karpowicz, P..

5 recordsLinked to original sources

bioRxiv2026

Membrane-dependent structural organization of cowpox virus CPXV012 and its recognition of TAP

Cowpox virus CPXV012 inhibits MHC class I antigen presentation by interfering with TAP-dependent peptide transport, but its membrane-dependent structural organization and dynamic behavior remain incompletely defined. Here, we investigated the conformational properties of CPXV012 in membrane-mimicking environments and in a model of the CPXV012-TAP complex. CPXV012 was divided into three peptide constructs corresponding to the N-terminal cytosolic region, transmembrane segment, and C-terminal ER-luminal domain. The peptides were analyzed by circular dichroism spectroscopy, multidimensional NMR spectroscopy, and molecular dynamics simulations, and the resulting structural information was integrated into a full-length CPXV012 model. CD spectra showed that CPX-E1 and CPX-C2 are predominantly disordered in aqueous solution but acquire ordered, mainly -helical features in DPC micelles. NMR analysis in DPC-d38 micelles provided residue-level assignments and structural restraints supporting restrained structure calculations for both peptides. In three independent 1 {micro}s molecular dynamics simulations of the CPXV012-TAP complex, CPXV012 preserved a reproducible two-helical organization. The N-terminal/transmembrane region behaved as a relatively stable structural element, whereas the ER-luminal segment showed greater local flexibility. Interface analysis indicated that CPXV012 contacts both TAP1 and TAP2, with recurrent interactions concentrated in the luminal Y47-I69 region and involving polar and charge-complementary contacts. These results support a model in which membrane-associated structuring positions CPXV012 for TAP recognition, while the flexible ER-luminal region forms the main TAP-interacting surface. This structural framework complements existing functional models of CPXV012-mediated TAP inhibition.

biochemistry↗

bioRxiv2026

Epithelial function of the circadian clock gene, Bmal1, in regulating the mucosa.

Circadian rhythms, 24-hour repeating oscillations in daily physiology, are implicated in maintaining intestinal homeostasis. These rhythms are driven by the circadian clock, a molecular timekeeper found throughout cells of the body, including those of the intestinal epithelium. Loss of clock function has been found to worsen colitis; however, it is not clear how the clock impacts regeneration which enables a tissue to return to its homeostatic set point following an injury. To investigate these questions, we used a conditional knockout of the core clock gene, Bmal1, in mouse colon epithelial cells. Our data show that prior to injury Bmal1 promotes colon mucus production, which increases in thickness and within goblet cells when mice are active and begin feeding. Bmal1 loss lowers mucus production but does not drive an apparent tissue phenotype until the system is injured and regenerates itself. In this context, Bmal1 epithelial loss drives a male-specific colitis phenotype and a delay in the ability of colon epithelial cells of both male and female mice to resolve injury to return to their homeostatic set point. Our data suggest that epithelial sex-specific clock rhythms are needed for optimal colon barrier homeostasis.

physiology↗

bioRxiv2025

Chrono-atlas of cell-type specific daily gene expression rhythms in the regenerating colon

The circadian clock is a molecular timer present throughout the body, including the gastrointestinal tract, where it regulates daily rhythms in physiology through the timing of rhythmic gene expression. Dysfunctional rhythms, caused by loss of clock timing and/or environmental disruption is implicated with gastrointestinal dysfunction and pathology. The large intestine (colon) is composed of many different types of cells with distinct gene expression programs and functions. How daily rhythms in transcript abundance are coordinated in the intestine at a cell-specific level is not known. Using single cell transcriptomics, we analyzed 24-hour gene expression in all major cell types of the proximal and distal regions of the colon following injury. We find that daily gene expression is not uniform: rhythmic genes, including circadian clock components, clock targets, and systemic programs, differ in their timing and are cell-type specific. Cells of the epithelium, stroma, and immune system to display strong rhythms in metabolic, protein processing, and stress response genes during regeneration. While stromal and muscle cells exhibit robust circadian clock gene rhythms irrespective of injury, epithelial cells show weaker clock oscillations that become 12-hours antiphasic during regeneration. These data, completed with smFISH validation, reveal an unexpected complexity to daily transcript levels in the colon, and provide a resource for future studies by identifying the cellular source of 24-hour transcript rhythms.

physiology↗

bioRxiv2024

Development of multi-species qPCR assays for a stress transcriptional profiling (STP) Chip to assess the resilience of salmonids to changing environments

Ecologically and socio-economically important salmonid fishes in Canada are threatened by diverse environmental stressors. However, predicting species responses to environmental change requires understanding the underlying molecular mechanisms governing environmental stress tolerance. Developing advanced molecular genetic tools will provide opportunities to predict how salmonid fishes will respond to environmental stressors and assess their adaptive potential and vulnerability into the future. Here, we developed a panel of Taqman quantitative PCR (qPCR) assays designed to measure mRNA transcript abundance at selected candidate loci for use across salmonids. We designed and applied those assays for use in a high-throughput nanofluidic OpenArray Stress Transcriptional Profiling Chip (STP-Chip) capable of 2688 simultaneous qPCR at multiple gene loci (112 targets for 12 samples in duplicate). Using the nanofluidic STP-Chip, we tested these 112 multi-species qPCR assays using gill, liver and muscle tissue from eight species of salmonids across four genera. Of the selected 112 assays, 69 assays showed amplification in gill, 64 in liver, and 67 in muscle across all eight salmonid species. The percentage of assays that showed amplification across three tissues varied between genera: In general, Salmo, Oncorhynchus, and Salvelinus species showed a higher success rate than Coregonus species. Stress, circadian rhythm, apoptosis, growth-metabolism, and detoxification-relevant assays showed high success rates for amplification across all salmonid species for all three tissues. In contrast, neural plasticity, appetite regulation, osmoregulation, immune function, endocrine disruption, and hypoxia-relevant assays showed low success. Not surprisingly, we observed tissue-specific variation among qPCR amplification patterns. There were significant differences in mRNA transcript abundance among species across the four genera, but we did not see variation between species from the same genus. These qPCR assays can be used to design custom STP-Chips that can be used for quantifying stress in salmonid fish, improving health through more accurate diagnostic tests for disease, and monitoring adaptation to accelerated climate change regionally and globally.

genomics↗

bioRxiv2023

The Emergence of Circadian Timekeeping in the Intestine

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-hour period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in ecdysone and Bursicon hormone signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

developmental biology↗