Biology subjects

Oakhill, J. S.

Publications and source records attributed to Oakhill, J. S..

14 recordsLinked to original sources

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↗

bioRxiv2026

Fanconi Anaemia E3 Ligase complex activity is regulated by a druggable metabolite binding site in FANCX

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

cancer biology↗

bioRxiv2025

Long-read sequencing-based atlas of tissue-specific expression of Drp1 transcript variants

Dynamin-related protein 1 (Drp1), encoded by DNM1L, is essential for mitochondrial fission, but its functional roles remain unclear due to isoform-specific effects from alternative splicing. Short-read RNA sequencing fails to resolve full-length isoforms involving distant exons, limiting our understanding. Here, we applied targeted long-read sequencing to profile full-length DNM1L transcripts in human left ventricle and iPSC-derived cardiomyocytes, recovering all annotated isoforms with conserved expression patterns and isoforms 1-4 being most abundant. Functional assays revealed that isoform abundance does not predict enzymatic activity. Extending this to six mouse tissues, we identified distinct, tissue-enriched expression profiles. Functional rescue in Drp1-knockout mouse embryonic fibroblasts showed isoform-dependent differences in mitochondrial fission. Isoforms lacking the A-insert (e.g., b and d) robustly rescued fission, while isoforms enriched in brain or muscle showed only partial rescue, suggesting exons 2 and 3 negatively regulate Drp1 activity. Our cross-species atlas integrates long-read transcriptomics with functional validation, revealing how isoform diversity underpins tissue-specific mitochondrial dynamics and physiological roles of Drp1. SummaryUsing long-read sequencing, we mapped full-length DNM1L/Dnm1l isoforms in human and mouse tissues, uncovering tissue-specific expression and isoform-dependent mitochondrial fission activity. This reveals how alternative splicing shapes Drp1 function, with implications for understanding its role in health and disease.

cell biology↗

bioRxiv2024

Modern Insights into Muscle Glycogen Phosphorylase Activity

Recent identification of new human muscle glycogen phosphorylation sites has renewed interest in understanding human variations in the regulation of glycogen metabolism and glucose homeostasis. This paper presents a detailed method for the measurement of glycogen phosphorylase (GPh) activity in skeletal muscle. Our approach incorporates modifications to existing radiolabelling assays, optimizing specificity and sensitivity while enabling the assessment of both active and total enzyme activity levels. The utilization of radioisotope tracers and scintillation counting ensures accurate quantification of GPh activity, which we use to validate a previously published reduction in GPh activity in an Actn3 deficient mouse model. Moreover, we introduce a step-by-step guide for data acquisition, highlight the use of appropriate homogenization, discuss the need for allosteric activators/inhibitors and the importance of assay optimization to record a GPh activity assay for skeletal muscle. In conclusion, our refined method not only contributes to a deeper understanding of glycogen metabolism in muscle tissue but also provides a framework for future investigations, underscoring its role in advancing research on glycogen utilization and glucose homeostasis. NEW & NOTEWORTHYThe study optimizes the glycogen phosphorylase radiolabelled activity assay, unveiling nuances in muscle homogenization, sample dilution, and caffeine inclusion. The research introduces standardized conditions, enhancing assay reliability and reproducibility across mouse strains to reveal sex specific variations in GPh activity and underscore novel distinctions in an Actn3 deficient mouse model. These findings advance our understanding of muscle glycogen metabolism, offering a crucial tool for researchers and facilitating meaningful inter-laboratory comparisons.

biochemistry↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Systemic ablation of Camkk2 impairs metastatic colonization and improves insulin sensitivity in TRAMP mice: Evidence for cancer cell-extrinsic CAMKK2 functions in prostate cancer

Despite early studies linking calcium-calmodulin protein kinase kinase 2 (CAMKK2) to prostate cancer cell migration and invasion, the role of CAMKK2 in metastasis in vivo remains unclear. Moreover, while CAMKK2 is known to regulate systemic metabolism, whether CAMKK2s effects on whole body metabolism would impact prostate cancer progression and/or related comorbidities is not known. Here, we demonstrate that germline ablation of Camkk2 slows, but does not stop, primary prostate tumorigenesis in the TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) genetic mouse model. Consistent with prior epidemiological reports supporting a link between obesity and prostate cancer aggressiveness, TRAMP mice fed a high-fat diet exhibited a pronounced increase in the colonization of lung metastases. We demonstrated that this effect on metastatic spread was dependent on CAMKK2. Notably, diet-induced lung metastases exhibited a highly aggressive neuroendocrine phenotype. Concurrently, Camkk2 deletion improved insulin sensitivity in the same mice. Histological analyses revealed that cancer cells were smaller in the TRAMP;Camkk2-/- mice compared to TRAMP;Camkk2+/+ controls. Given the differences in circulating insulin levels, a known regulator of cell growth, we hypothesized that systemic CAMKK2 could promote prostate cancer cell growth and disease progression in part through cancer cell-extrinsic mechanisms. Accordingly, host deletion of Camkk2 impaired the growth of syngeneic murine prostate tumors in vivo, confirming nonautonomous roles for CAMKK2 in prostate cancer. Cancer cell size and mTOR signaling was diminished in tumors propagated in Camkk2-null mice. Together, these data indicate that, in addition to cancer cell-intrinsic roles, CAMKK2 promotes prostate cancer progression via tumor-extrinsic mechanisms. Further, we propose that CAMKK2 inhibition may also help combat common metabolic comorbidities in men with advanced prostate cancer.

cancer biology↗

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

Elevated Neuropeptide Y1 Receptor Signaling Contributes to β-cell Dysfunction and Failure in Type 2 Diabetes

Loss of functional {beta}-cell mass is a key factor contributing to the poor glycaemic control in type 2 diabetes. However, therapies that directly target these underlying processes remains lacking. Here we demonstrate that gene expression of neuropeptide Y1 receptor and its ligand, neuropeptide Y, was significantly upregulated in human islets from subjects with type 2 diabetes. Importantly, the reduced insulin secretion in type 2 diabetes was associated with increased neuropeptide Y and Y1 receptor expression in human islets. Consistently, pharmacological inhibition of Y1 receptors by BIBO3304 significantly protected {beta}-cells from dysfunction and death under multiple diabetogenic conditions in islets. In a preclinical study, Y1 receptor antagonist BIBO3304 treatment improved {beta}-cell function and preserved functional {beta}-cell mass, thereby resulting in better glycaemic control in both high-fat-diet/multiple low-dose streptozotocin- and db/db type 2 diabetic mice. Collectively, our results uncovered a novel causal link of increased islet NPY-Y1 receptor signaling to {beta}-cell dysfunction and failure in human type 2 diabetes. These results further demonstrate that inhibition of Y1 receptor by BIBO3304 represents a novel and effective {beta}-cell protective therapy for improving functional {beta}-cell mass and glycaemic control in type 2 diabetes.

cell biology↗

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↗

bioRxiv2020

In depth analysis of kinase cross screening data to identify CAMKK2 inhibitory scaffolds

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays a central role in many cell signaling pathways. CAMKK2 activates CAMK1, CAMK4, AMPK, and AKT leading to numerous physiological responses. Deregulation of CAMKK2 is linked to several diseases, suggesting utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work we review the role of CAMKK2 in biology and disease. Through analysis of literature and public databases we have identified starting points for CAMKK2 inhibitor medicinal chemistry campaigns. These starting points provide an opportunity for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

cancer biology↗

bioRxiv2018

Meteorin-like (Metrnl) adipomyokine improves glucose tolerance in type 2 diabetes via AMPK pathway

Meteorin-like (metrnl) is a recently identified adipomyokine that has beneficial effects on glucose metabolism. However, its underlying mechanism of action is not completely understood. In this study, we have shown that a level of metrnl increase in vitro under electrical-pulse-stimulation (EPS) and in vivo in exercise mice, suggesting that metrnl is an exercise-induced myokine. In addition, metrnl increases glucose uptake through the calcium-dependent AMPK pathway. Metrnl also increases the phosphorylation of HDAC5, a transcriptional repressor of GLUT4, in an AMPK-dependent manner. Phosphorylated HDAC5 interacts with 14-3-3 proteins and sequesters them in the cytoplasm, resulting in the activation of GLUT4 transcription. The intraperitoneal injection of recombinant metrnl improves glucose tolerance in mice with high fat-induced obesity or type 2 diabetes (db/db), but this is not seen in AMPK {beta}1{beta}2 muscle-specific null mice (AMPK {beta}1{beta}2 MKO). In conclusion, we have demonstrated that metrnl induces beneficial effects on glucose metabolism via AMPK and is a promising therapeutic candidate for glucose-related diseases such as type 2 diabetes.

biochemistry↗

bioRxiv2018

DNA-PKcs-mediated phosphorylation of AMPKγ1 regulates lysosomal AMPK activation by LKB1

Autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed DNA-PKcs upstream of the AMP-dependent protein kinase (AMPK) and ULK1 kinase. In normal culture conditions, DNA-PKcs interacted with AMPK and phosphorylated its nucleotide-sensing {gamma}{gamma}1 subunit at Ser-192 and Thr-284, both events being significantly reduced upon AMPK activation. Alanine substitutions of DNA-PKcs phosphorylation sites in AMPK{gamma}{gamma}1 reduced AMPK activation without affecting its nucleotide sensing capacity. Instead, the disturbance of DNA-PKcs-mediated phosphorylation of AMPK{gamma}{gamma} inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with LKB1. Taken together, our data suggest that DNA-PKcs-mediated phosphorylation of AMPK{gamma}{gamma} primes AMPK complex to the lysosomal activation by LKB1 thereby linking DNA damage response to autophagy and cellular metabolism.

cell biology↗

bioRxiv2018

Increased autophagy in ephrinB2 deficient osteocytes is associated with hypermineralized, brittle bones

Mineralized bone forms when collagen-containing osteoid accrues hydroxyapatite crystals. This process has two phases: a rapid initiation (primary mineralization), followed by slower accrual of mineral (secondary mineralization) that continues until that portion of bone is renewed by remodelling. Within the bone matrix is an interconnected network of cells termed osteocytes. These cells are derived from bone-forming osteoblasts. Osteoblast differentiation requires expression of ephrinB2, and we were intrigued about why ephrinB2 continues to be expressed in mature osteocytes. To determine its function in osteocytes, we developed an osteocyte-specific ephrinB2 null mouse and found they exhibited a brittle bone phenotype. This was not caused by a change in bone mass, but by an intrinsic defect in the strength of the bone material. Although the initiation of osteoid mineralization occurred at a normal rate, the process of secondary mineralization was accelerated in these mice. The maturing mineralized bone matrix incorporated mineral and carbonate more rapidly than controls, indicating that osteocytic ephrinB2 suppresses mineral accumulation in bone. No known regulators of mineralization were modified in the bone of these mice. However, RNA sequencing showed differential expression of a group of autophagy-associated genes, and increased autophagic flux was confirmed in ephrinB2 knockdown osteocytes. This indicates that the process of secondary mineralization in bone makes use of autophagic machinery in a manner that is limited by ephrinB2 in osteocytes, and that this process may be disrupted in conditions of bone fragility.

cell biology↗