Biology subjects

Ovens, A. J.

Publications and source records attributed to Ovens, A. J..

5 recordsLinked to original sources

bioRxiv2026

Leukocyte Immunoglobulin-Like Receptor B1 and its Interactions with Human Leukocyte Antigens

Interactions between Human Leukocyte Antigen (HLA) molecules and their cognate immunoreceptors are essential for regulating innate and adaptive immune cell functions. Leukocyte Immunoglobulin-like Receptors (LILRs) are key regulators of HLA-mediated immune responses, owing to their broad expression across immune cell populations and their ability to modulate both immune activation and tolerance. Among these, LILRB1-HLA interactions are increasingly recognised as important in transplantation, chronic infection and cancer therapies. Unlike other HLA-binding receptors, which recognise epitopes specific to HLA subsets, LILRB1 primarily engages the relatively conserved 3 and {beta}2-microglobulin components of HLA molecules, supporting its role as a broad regulator of pan-HLA class I-mediated functions. Nonetheless, there have been conflicting findings regarding the breadth of LILRB1-HLA-I interactions. While direct affinity studies on a limited subset of HLA-I molecules have revealed no significant differences in LILRB1 binding, broader analyses using single-antigen bead arrays suggest underlying variability. Here, we show through a broad binding assay that, while LILRB1 is a broad HLA-I-binding receptor, it exhibits differential preferences across HLA-I allotypes. Molecular dynamics analyses of the HLA-I-LILRB1 interface suggest that HLA-3 domain dynamism underlies these binding differences. We further determined the crystal structure of LILRB1 and used it to highlight intrinsic structural flexibility within its domains. Finally, these structural insights were leveraged to refine our understanding of the binding modalities of therapeutic monoclonal antibodies currently described. Together, our findings establish structural and mechanistic bases for differential HLA-I recognition by LILRB1 and provide insights into immunotherapeutic targeting of LILRB1.

immunology↗

bioRxiv2026

Fine tuning energy metabolism in skeletal muscle: Discovery of a novel autoinhibitory mechanism in the N-terminal extension of AMPKγ3

AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an {beta}{gamma} heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the 2{beta}2{gamma}3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within {gamma}3 subunit. Deletion of the {gamma}3-NTE from 2{beta}2{gamma}3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the {gamma}3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the {gamma}3-NTE (residues 129-168), that directly interacts with the C-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a {gamma}3-NTE -helix ({gamma}3-iHelix; [~]T142-E154) and the C-helix in the 2{beta}2{gamma}3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the {gamma}3 autoinhibition.

biochemistry↗

bioRxiv2022

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers. HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5. C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade. C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma. C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Novel torin1-sensitive phosphorylation sites on the metabolic regulator AMPK revealed by label-free mass spectrometry

AMPK and mTORC1 are nutrient-sensitive protein kinases that form a fundamental negative feedback loop that governs cell growth and proliferation. AMPK is an {beta}{gamma} heterotrimer that is directly phosphorylated by mTORC1 on 2S345 to suppress AMPK activity and promote cell proliferation under nutrient stress conditions. Using mass spectrometry, we generated precise phosphorylation profiles of all 12 AMPK complexes expressed in proliferating human cells. Of the 18 phosphorylation sites detected, seven were sensitive to pharmacological mTORC1 inhibition, including four in the AMPK {gamma}2 isoform NH2-terminal domain and 2S377 which is located in the nucleotide-sensing motif. In particular, {beta}1S182 and {beta}2S184 were found to be mTORC1 substrates in vitro and near-maximally or substantially phosphorylated under cellular growth conditions. {beta}S182 phosphorylation was elevated in 1-containing complexes, relative to 2, an effect partly attributable to the non-conserved -subunit serine/threonine-rich loop. While mutation of {beta}1S182 to a non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity, {beta}2-S184A mutation increased nuclear AMPK activity and enhanced cell proliferation under nutrient stress. We conclude that mTORC1 governs the nuclear activity of AMPK to regulate transcription factors involved in metabolism and cell survival during nutrient shortage.

biochemistry↗

bioRxiv2021

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPK{gamma}3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPK{gamma}3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if {gamma}3 plays a role in adipose thermogenesis and browning. Methods: Global AMPK{gamma}3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in {gamma}3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of {gamma} subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the and {beta} subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a {beta}3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function. Results: Genetic ablation of {gamma}3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. {gamma}3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in {gamma}3 KO mice showed a partial loss of AMPK2 activity, which was associated with reduced levels of AMPK2 and {beta}2 subunit isoforms. Notably, {gamma}3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected {gamma}3 in BAT and found that it preferentially interacts with 2 and {beta}2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and {gamma}3 KO mice. Conclusions: These results demonstrate that {gamma}3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that {gamma}3 is dispensable for thermogenesis and browning of iWAT.

physiology↗