Biology subjects

Allen, K. N.

Publications and source records attributed to Allen, K. N..

14 recordsLinked to original sources

bioRxiv2026

Iron export and lipid droplets shield deep-diving elephant seal cells from lipid peroxidation

Elephant seals are remarkable breath-hold divers, capable of remaining submerged for up to two hours during diving bouts. These dives entail repeated, extreme hypoxia/reoxygenation events that would induce severe lipid peroxidation and tissue dysfunction in most mammals. Here, we show that primary vascular endothelial cells derived from elephant seals possess an intrinsic resistance to lipid peroxidation. Comparative transcriptomic and lipidomic profiling across seal, human, and sheep cells identified ferroptosis - an iron-dependent, lipid peroxidation-driven cell death pathway - as uniquely regulated in seal cells following hydroperoxide exposure. Mechanistically, seal cells exhibit robust baseline expression of acyl-CoA synthetase long-chain family member 3 (ACSL3), alongside rapid, seal-specific induction of the sole mammalian iron exporter, ferroportin (SLC40A1). Functional validation using genetic and pharmacological approaches revealed that seal cells are naturally enriched in monounsaturated fatty acids and triglycerides and utilize lipid droplet biogenesis and active iron export as dual protective axes to evade lipid peroxidation. Together, these findings show that elephant seal cells employ a coordinated cytoprotective network of lipid remodeling and iron handling to withstand the severe challenges of deep diving. SIGNIFICANCE STATEMENTDeep-diving marine mammals repeatedly experience extreme hypoxia-reoxygenation events that would induce severe oxidative damage in most terrestrial mammals. However, vascular cells derived from seals naturally resist lipid peroxidation, a major driver of ischemia-reperfusion injury. Here, we show that elephant seal endothelial cells evade lipid peroxidation through two complementary mechanisms: lipid droplets that sequester peroxidation-prone phospholipids, and rapid iron export that limits lipid peroxide formation. These findings reveal naturally evolved cellular strategies that protect against vascular oxidative stress, offering new insights into physiological resilience against ischemia-reperfusion injury.

physiology↗

bioRxiv2026

Deciphering AMP deaminase-2 structure, activators and regulators underpinning cellular function in human fructose and nucleotide metabolism

AMP deaminase (AMPD) plays an integral role in fructose metabolism via its regulation by ATP, GTP and phosphate (Pi). The fructose catabolic pathway consumes ATP, producing ADP, which is further metabolized to AMP, triggering a cascade of reactions initiated by AMPD. This degradative pathway results in the final product uric acid, which is associated with metabolic acidosis, mitochondrial dysfunction, and gout. Understanding the regulation of the human liver AMPD isozyme (hAMPD2-2) under physiological conditions and under fructose consumption conditions will enable the design of targeted therapeutics to block the accumulation of uric acid. We report the first successful expression and purification from Escherichia coli of both the full-length and catalytic domains of hAMPD2-2. Steady-state kinetics confirmed allosteric activation by ATP of both the full-length and catalytic domains of hAMPD2-2 at physiological ATP concentrations (2-5 mM), suggesting that the allosteric ATP-binding site is located in the catalytic domain. Competitive inhibition by GTP of the ATP-activated enzyme, with Ki values of 74 and 101 M for the full-length and catalytic domains, respectively, was also consistent with this regulatory model. Pi, previously described in yeast AMPD as a competitive inhibitor, was shown to play a more nuanced role, that of enhancing inhibition of hAMPD2-2 when the enzyme is complexed to GTP, via competition at the ATP allosteric site. Pi binding thus further inhibits the pathway under normal physiological conditions, limiting production of cellular uric acid unless and until Pi and GTP levels are low.

biochemistry↗

bioRxiv2026

Glycoconjugate diversification in Campylobacter concisus is determined by two glycosyltransferases

Bacterial glycoconjugates are structurally diverse, with enormous variation in sugar identity, modifications and linkages. Glycoconjugates play key roles in numerous cell functions, acting as the primary interface with the environment. Asparagine (N)-linked glycosylation has been extensively studied in the pathogenic Campylobacter genus, due to the availability of numerous genome sequences and the highly conserved pathway logic, despite the final N-linked glycan product diversity. We recently reported on a partitioning of N-linked glycan structures between the Campylobacter species, focused on the inclusion of a C6-carboxyl-sugar in the third position of the growing glycan in Campylobacter concisus. However, at the time, the final glycan was not fully defined. Here, we identify the final glycan product in C. concisus, demonstrating surprising substrate promiscuity of the GT-A enzyme, PglI, which adds the penultimate sugar, and uncover a previously uncharacterized enzyme (GT-25) that unexpectedly adds the final sugar to complete the heptasaccharide product. Through a detailed study of these two C. concisus pathway enzymes, the intermediate and final glycans were defined, with determination of linkage positions of major and minor isomeric products following each glycan addition, through high-resolution electronic excitation dissociation tandem mass spectrometry. These findings on the Group II C. concisus N-linked glycan highlight the diversification of the glycan and the utilization, at the non-reducing end, of GlcNAc over GalNAc, which is dominant in the Group I species.

biochemistry↗

bioRxiv2026

A nicotine biosensor derived from microbial screening

Physiologically relevant biosensors are in increasingly high demand, yet existing ones are severely limited in the number and type of biomarkers that are detected. The lack of biorecognition elements for most medically relevant biomarkers restricts the development of next generation single and continuous use monitors. Over billions of years, microbes have evolved a vast array of proteins to sense and metabolize small molecules, including those pertinent to human health. Of particular interest to us is the identification and subsequent integration of new microbial redox enzymes into electronic biosensors building off the established electrochemical technology of the continuous glucose monitor. Here we deploy genomic screening to identify analyte specific redox enzymes for biosensor development. As a proof of concept, we report the first electrochemical enzyme-based nicotine biosensor from a novel microbial enzyme, and use a variant with improved catalytic performance to enhance sensor performance. The biosensor detects nicotine over 0.4-100 M, a range relevant to nicotine concentrations present in active smoker sweat, saliva, gastric juice, and urine. This microbial mining approach for discovering redox enzymes expands the sensing parts toolbox available over conventional antibodies and aptamers.

bioengineering↗

bioRxiv2026

Phthalate exposure induces inflammatory signaling and alters mitochondrial respiration in marine mammal and human cells

This study investigated the transcriptional and bioenergetic responses to monoethylhexyl phthalate (MEHP) in primary fibroblasts derived from northern elephant seals (Mirounga angustirostris), common dolphins (Delphinus delphis), and humans, using RNA-seq, extracellular flux assays, and high-resolution microscopy of the mitochondrial reticulum. MEHP exposure did not induce cytotoxicity but triggered species-specific changes in gene expression and mitochondrial metabolism and morphology. Human cells showed the greatest transcriptional response, upregulating genes involved in detoxification, antioxidant, and inflammation while downregulating lipid metabolism pathways. The highest dose also decreased mitochondrial respiration and increased mitochondrial fragmentation, triggering a metabolic shift toward glycolysis. Elephant seal cells showed delayed glycolytic shifts, maintaining mitochondrial respiration and upregulating antioxidant, immune, and metabolic pathway genes through the highest dose. Despite mitochondrial fragmentation, they upregulated mitochondrial fusion/fission and trafficking genes. Dolphin cells exhibited the fewest changes in gene expression, mostly in hormone signaling and mitotic pathways. They showed dose-dependent declines in both respiration and glycolytic rates, even at the lowest concentration, yet maintained mitochondrial structural integrity while upregulating stress- and hypoxia-induced genes. These distinct strategies highlight species-specific susceptibility to toxicant-induced stress, offering new insights into how marine mammals respond to plastic-derived contaminants and reinforcing the need for species-specific ecotoxicological risk assessments.

physiology↗

bioRxiv2025

Detergent exchange from lipid nanoparticles into detergent micelles unlocks a tool for biochemical and kinetic characterization of membrane proteins

Bacterial membrane proteins make up [~]30% of the prokaryotic genome and play key roles in infection and virulence. Membrane protein chemistry has advanced in recent years, including purification strategies that mimic "native-like" lipid environments, such as lipid nanoparticles, amphipols, and nanodiscs. The use of styrene maleic acid co-polymers to form a lipid nanoparticle has become increasingly common in membrane protein purification, especially for proteins which are not amenable to detergent extraction from the cellular membrane fraction. Yet, for some biochemical and biophysical methods it is preferable to use detergent-solubilized protein. Here we show a general exchange method to transfer membrane proteins from lipid nanoparticles to detergent micelles while retaining protein fold, homogeneity and function. Conditions were first optimized for co-polymer dispersion and recovery into detergents, and analytical methods employed to assess activity and quality of detergent-solubilized proteins. Twelve protein targets were purified in co-polymer based on a 16-polymer screen. This selection was followed by an eight-detergent screen in the presence of calcium ions for optimal dissolution of the nanoparticle, producing detergent-stabilized protein. In all membrane proteins assessed, homogeneity and folding were retained from the initial purification in lipid nanoparticles through the detergent-exchange protocol. For membrane enzymes that have proven to be experimentally intractable once detergent solubilized, we were able to observe catalytic activity using the detergent-exchanged material. The use of this protocol to purify membrane proteins provides great versatility for biochemical and kinetic characterization than was previously accessible.

biochemistry↗

bioRxiv2025

Wearable sweat nicotine biosensor based on a nicotineoxidoreductase and its natural electron acceptor

The advent of wearable biosensors is empowering clinical and lifestyle decision making. Nicotine is a drug of significant negative impact on public health given the prevalence of smoking and vaping. Yet noninvasive, continuous monitoring of nicotine is challenging due to lack of specific and sensitive biosensing elements. Here, we report a wearable highly sensitive nicotine biosensor and demonstrate on-body deployment of the sensor in a first-in-human study. The biosensor comprises nicotine oxidoreductase (NicA2) with its natural cytochrome c electron acceptor (CycN) as mediator: both proteins play a key role in the nicotine-degrading bacterium, Pseudomonas putida S16. The biosensor detects nicotine over four-orders of magnitude (0.1-100 M) with nanomolar sensitivity (LOD [~]33.6 nM), performs in the physiological pH range (pH 6-9), and is highly selective against common interferants and the major human nicotine metabolite, cotinine. Incorporation of the biosensor in a custom wearable device enables real-time measurement of nicotine from locally induced sweat on volunteer subjects and demonstrates higher analytical accuracy than gold-standard mass spectrometry.

bioengineering↗

bioRxiv2025

Selection of nanobodies against liponanoparticle-embedded membrane proteins by yeast surface display

Single-domain antibodies, known as nanobodies (Nbs), are widely used in structural biology, therapeutics, and as molecular probes in biology and biotechnology. Nbs towards soluble proteins are routinely developed via alpaca immunization or directed evolution in yeast cell-surface display. However, for membrane proteins, the targets are generally detergent-solubilized, and there remains a need for Nb development methods against membrane proteins in a native-like membrane environment. To address this need, we present a protocol for Nb selection via extraction of membrane proteins into amphiphilic polymers such as styrene-maleic acid to produce purified membrane proteins in stable liponanoparticles. Proof of generality is demonstrated by applying the pipeline to four membrane-resident enzymes of differing fold, oligomerization state, and membrane topology (reentrant membrane helix, transmembrane, membrane-associated). Following screening for optimal stabilization into liponanoparticles, Nbs were selected against four target proteins from glycoconjugate biosynthesis pathways. The selected Nbs showed high affinity and selectivity towards their target proteins with KD apparent values ranging from 15 nM to 200 nM, depending on the Nb-protein conjugate. In accordance with their tight binding, various Nb-protein complexes were found to be stable to size-exclusion chromatography purification. The Nbs were also amenable to sortase-mediated ligation, enabling their conversion into molecular probes for the target membrane protein. The ability to select for such high-affinity Nb against membrane proteins in SMALP will facilitate their widespread application in cell biology and biomedical applications.

biochemistry↗

bioRxiv2024

Structural Basis for the Improved RNA-Clamping of Amidino-Rocaglates to eIF4A1

Eukaryotic Initiation Factor 4A-1 (eIF4A1) is an ATP-dependent RNA helicase that unwinds 5-UTR mRNA secondary structures to facilitate cap-dependent translation initiation. Rocaglates, a class of natural products typified by rocaglamide A (RocA), possess anti-neoplastic and anti-infectious activity mediated by their interaction with eIF4A1. Rocaglates inhibit cap-dependent translation initiation by "clamping" eIF4A1 onto polypurine RNA, impeding ribosome scanning. A novel class of rocaglate derivatives, amidino-rocaglates (ADRs) which feature an amidine ring fused to the rocaglate core, are particularly effective at promoting eIF4A1-RNA-clamping compared to other rocaglate congeners. Herein, we present the X-ray crystal structure of an ADR in complex with eIF4A1, the nonhydrolyzable ATP ground-state mimic adenylyl-imidodiphosphate (AMPPNP), and poly r(AG)5 RNA refined to 1.69 [A] resolution. The binding pose and interactions of the ADR with eIF4A1 do not differ substantially from those of RocA, prompting investigation of the basis for enhanced target engagement. Computational modeling suggests that the rigidified ADR scaffold is inherently preorganized in an eIF4A1-RNA-binding-competent conformation, thereby avoiding entropic penalties associated with RocA binding. This study illustrates how conformational rigidification of the rocaglate scaffold can be leveraged to improve potency for the development of rocaglates as potential anticancer and anti-infectious agents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/616674v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@3c6dcorg.highwire.dtl.DTLVardef@1f4ae24org.highwire.dtl.DTLVardef@d4e264org.highwire.dtl.DTLVardef@5b1bde_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Functionally Validated Proteome-Wide Bioinformatic Annotation of the Monotopic Phosphoglycosyl Transferase Family

Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives. SignificanceUncovering the function and specificity of enzymes responsible for glycoconjugate biosynthesis traditionally requires a multi-faceted and individually curated approach. This is especially true for bacterial glycoconjugates due to greater monosaccharide diversity and a paucity of established structural information. Here we leverage bioinformatic and in-vitro tools to predict and validate substrate specificity for a unique, exclusively bacterial family of enzymes responsible for the first step in many of these glycan assembly pathways. We further show that this platform is suitable for enhanced functional annotation and inhibitor testing, paving the way for the development of urgently needed antibiotics.

biochemistry↗

bioRxiv2023

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-{kappa}B pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of I{kappa}B kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-{kappa}B pathway by acting as a mediator of conformational changes in NEMO.

molecular biology↗

bioRxiv2023

Synergistic computational and experimental studies of a phosphoglycosyltransferase membrane/ligand ensemble

Complex glycans serve important functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates, such as un-decaprenol phosphate in bacteria. In this study we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018, as a detergent solubilized construct. PglC is a monotopic phosphoglycosyl transferase (PGT), that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results reveal a specific interaction of undecaprenol phosphate with PGT cationic residues and suggest a role for critical conformational transitions and electrostatic steering in substrate recognition, overcoming significant energetic barriers to binding. The study highlights that computation, guided by fundamental chemical principles, can advance the study of biochemical processes at membrane bilayers and provide chemical insight at a molecular level that cannot be derived by experiment alone. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/539694v1_ufig1.gif" ALT="Figure 1"> View larger version (100K): org.highwire.dtl.DTLVardef@19a38d9org.highwire.dtl.DTLVardef@a41d8org.highwire.dtl.DTLVardef@168e48borg.highwire.dtl.DTLVardef@1749bd8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Protein-Metabolite Interactomics Reveals Novel Regulation of Carbohydrate Metabolism

Metabolism is highly interconnected and also has profound effects on other cellular processes. However, the interactions between metabolites and proteins that mediate this connectivity are frequently low affinity and difficult to discover, hampering our understanding of this important area of cellular biochemistry. Therefore, we developed the MIDAS platform, which can identify protein-metabolite interactions with great sensitivity. We analyzed 33 enzymes from central carbon metabolism and identified 830 protein-metabolite interactions that were mostly novel, but also included known regulators, substrates, products and their analogs. We validated previously unknown interactions, including two atomic-resolution structures of novel protein-metabolite complexes. We also found that both ATP and long-chain fatty acyl-CoAs inhibit lactate dehydrogenase A (LDHA), but not LDHB, at physiological concentrations in vitro. Treating cells with long-chain fatty acids caused a loss of pyruvate/lactate interconversion, but only in cells reliant on LDHA. We propose that these regulatory mechanisms are part of the metabolic connectivity that enables survival in an ever-changing nutrient environment, and that MIDAS enables a broader and deeper understanding of that network.

biochemistry↗

bioRxiv2021

Repeated stimulation of the HPA axis alters white blood cell counts without increasing oxidative stress or inflammatory cytokines in fasting elephant seal pups

The hypothalamic-pituitary-adrenal (HPA) axis controls the release of glucocorticoids, which regulate immune and inflammatory function by modulating cytokines, white blood cells (WBCs), and oxidative stress via glucocorticoid receptor (GR) signaling. Although the response to HPA activation is well characterized in many species, little is known about the impacts of HPA activation during extreme physiological conditions in marine mammals. Hence, we challenged 18 simultaneously fasting and developing elephant seal pups with daily intramuscular injections of adrenocorticotropin (ACTH), a GR antagonist (RU486), or a combination (ACTH+RU486) for four days (4d). We collected blood at baseline, two hours (2h), and 4d after the beginning of treatment. ACTH and ACTH+RU486 elevated serum aldosterone and cortisol at 2h, with effects diminishing at 4d. RU486 alone induced a compensatory increase in aldosterone, but not cortisol, at 4d. ACTH decreased neutrophils at 2h while decreasing lymphocytes and increasing neutrophil:lymphocyte ratio at 4d. These effects were abolished by RU486. Despite alterations in WBCs, there was no effect of ACTH or RU486 on transforming growth factor-{beta} or interleukin-6 levels; however, both cytokines decreased with the 4-d fasting progression. Similarly, ACTH did not impact protein oxidation, lipid peroxidation, or antioxidant enzymes, but plasma isoprostanes and catalase activity decreased while glutathione peroxidase increased with fasting progression. These data demonstrate differential acute (2h) and chronic (4d) modulatory effects of HPA activation on WBCs and that the chronic effect is mediated, at least in part, by GR. These results also underscore elephant seals resistance to potential oxidative stress derived from repeated HPA activation. Summary statementMany species experience oxidative stress and inflammation after repeated activation of the hypothalamic-pituitary-adrenal axis. We show that simultaneously fasting and developing elephant seals are resistant to repeated hypothalamic-pituitary-adrenal axis activation.

physiology↗