Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

{beta}-arrestins 1 and 2 are intracellular adaptor proteins that regulate the subcellular localisation and signalling of GPCRs including Parathyroid Hormone Receptor 1 (PTH1R), which produces {beta}-arrestin-dependent signalling that may selectively stimulate bone anabolism. {beta}-arrestins can themselves be regulated by post-translational modifications including phosphorylation, which dictates the translocation and signalling of the few GPCRs studied in this context to date. We aimed to investigate whether phosphorylation of {beta}-arrestins modulates their subcellular translocation in response to PTH1R activation. Translocation of {beta}-arrestins to the plasma membrane and early endosomes was quantified using BRET biosensors and immunocytochemistry with high content analysis. Phosphoknockout or phosphomimetic mutations were introduced to previously demonstrated phosphorylation sites in {beta}-arrestins 1 and 2, initially in bulk to assess overall effects. Plasma membrane translocation was altered only subtly, whereas early endosome translocation was markedly reduced for both mutations but to a greater degree for the phosphomimetic. Contributions of individual or paired sites were assessed by restoration to wildtype from the bulk phosphomimetic mutants. Dual restoration of S13 and T275 in {beta}-arrestin 1, or the analogous sites in {beta}-arrestin 2, restored the greatest degrees of early endosomal translocation, with complete restoration for {beta}-arrestin 1 and partial for {beta}-arrestin 2. Restoration of other sites had minimal or no effect. Dephosphorylation therefore appears to be required for PTH1R-stimulated {beta}-arrestin early endosomal translocation, with different phosphorylation sites implicated than some other GPCRs. These findings raise the possibility of differential {beta}-arrestin regulation by distinct stimuli which may have implications in therapeutic targeting of {beta}-arrestin-dependent pathways.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

The transport of about 70 enzymes to lysosomes depends on mannose 6-phosphate signals formed by GNPTAB. Editing of Gnptab in an intestinal mouse cell line revealed the loss of multiple lysosomal enzymes associated with the accumulation of sphingomyelins, ceramides, and cholesterol, and reduced levels of bis(monoacylglycero)phosphate (BMP) in lysosomal proteomes and lipidomes. By cross-correlation we identified two subsets of lysosomal lipid-modifying enzymes linked with mixed unsaturated or di-monosaturated BMP. Autophagy-related proteins functioning in early stages of autophagosome formation associated with neutral ceramides, whereas proteins involved in autophagosome-lysosome fusion correlated with negatively charged BMPs. Therefore, the high cholesterol and low BMP level of lysosomes might be causal for impaired autophagic flux in GNPTAB deficient cells. We propose a functional axis of three lysosomal proteins as potential target to improve the autophagic flux in cells with dysfunctional lysosomes and in a newly established intestinal organoid model suitable as novel experimental tool.

Background/Objectives: Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, mitigates cellular stress by detoxifying reactive aldehydes produced during alcohol exposure and endogenous metabolic processes. ALDH2*2 polymorphism, which results in deficient catalytic activity, is prevalent in East Asian populations and is associated with an increased risk of cancer and other diseases. While the effects of ALDH2 deficiency on cellular stress responses are well documented, the specific metabolic dysregulations arising from this deficiency require further investigation. Methods; An ALDH2 knockout subline of SKBR-3 cells was established using CRISPR/Cas9 strategy. The metabolomic profiles of control (SKBR-3/C) and ALDH2 knockout (SKBR-3/ADKO) cells were evaluated via ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Results: ALDH2 knockout in SKBR-3 cells resulted in increased cellular stress, evidenced by elevated levels of 8-OHdG, reactive oxygen species (ROS), and malondialdehyde (MDA). Metabolomic analysis of the paired cell lines assigned a total of 5,033 signals, of which 281 were significantly different (fold change [&ge;] 2, p-value <0.05) between the two groups. Pathway analysis with MetaboAnalyst identified significant metabolic pathways associated with ALDH2 deficiency, particularly those involving arginine biosynthesis, and arginine and proline metabolism. ALDH2 deficiency reprogramed arginine metabolism toward polyamine pathways while suppressing the urea cycle. Network analysis further revealed alterations in amino acid and lipid metabolism in ALDH2-deficient cells. Conclusions; This study provides a novel insight into the role of ALDH2 in cancer metabolism and may have broader implication for cancer biology.

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX < 10%) and high (IRX > 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B'', which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

Telomeres, the ends of linear chromosomes, form a cap composed of specific telomeric DNA associated with specialized proteins that ensures efficient protection against chromosome degradation and, consequently, overall genome instability. Three main complexes have been implicated in chromosome end protection, namely telomerase, shelterin, and the CST complex (CTC1/Cdc13-STN1-TEN1). In a recent study, we uncovered mutants of the S. cerevisiae CST in which damage was sensed both by the two major spindle checkpoints and the major DNA damage checkpoint. In the best-studied of these mutants, stn1-sz2, the stability and organization of the mitotic tubulin spindle was profoundly affected, as were the organization of the centrosomes and the kinetochores. Here, we find that the stn1-sz2 mutant is hypersensitive to deletion mutants of two members of the kinetochores, Ctf19 and Mcm21, which correlated with mis-localization of their associated subunit in the COMA complex, Okp1, during anaphase. Defects in interactions between the kinetochores and the tubulin spindle are entirely sensed by the SAC (spindle assembly checkpoint). Here, we find that some of the stn1-sz2 damage is recognized by the Mad2 checkpoint protein independently from the SAC. The data presented here contribute to a better understanding of the damage in stn1-sz2, the prototype of novel mutants of telomeric CST characterized by mitotic-spindle-damage in addition to the classical telomeric-DNA-damage.

Hearing loss and deafness as a result of hair cell loss cannot be restored due to the incapacity of spontaneous regeneration of these cells. Compounds that manipulate key signaling pathways can potentially regenerate cochlear hair cells. In order to test different compounds that promote hair cell regeneration in vitro models can be used. Three-dimensional cultures have allowed the expansion and experimentation of human and mouse inner ear organoids. This is mainly performed in the embryonic or the early postnatal developmental stage. However, since the majority of patients with hearing loss are adult, it is crucial to understand the adult inner ear regenerative capacity. Here, we evaluated progenitor cell markers in the adult human and mouse inner ear and the generation, expansion and differentiation of cochlear organoids derived from the adult human and mouse inner ear. Cochlear and vestibular sensory epithelium from adult humans; and from adult and aged mice express the progenitor markers SOX2 and LGR5; and can generate organoids in vitro. By optimizing the culture conditions, organoids derived from the cochlea and vestibular organ from adult human and adult and aged mouse differentiated to Myosin 7A positive cells. This indicates that the adult inner ear has regenerative capacity. These findings are encouraging for future regenerative therapies to cure hair cell-related hearing loss and deafness.

Nucleolar alterations are characteristic features of cancer cells; however, how changes in the overall morphology relate to internal subcompartment organization and ribosomal RNA (rRNA) dynamics remains unclear. In this study, the nucleolar organization of non-transformed MCF-10A mammary epithelial cells and MDA-MB-231 breast cancer cells was quantitatively compared. MDA-MB-231 cells exhibited differences in nucleolar morphology, accompanied by altered spatial organization of the dense fibrillar component and granular component, and accelerated intranucleolar trafficking of nascent rRNA. Pharmacological perturbation with SU056 shifted multiple structural and dynamic parameters toward an MCF-10A-like profile. Conversely, transient physicochemical perturbation with 1,6-hexanediol induced several MDA-MB-231-like nucleolar features in MCF-10A cells, which returned to their untreated profile after washout. These findings support the view that cancer-associated nucleolar alterations are an integrated reorganization across morphological, spatial, and dynamic dimensions. The observed reversibility further indicates that cancer-associated nucleolar organization can be remodeled in response to perturbations. By linking the morphology, subcompartment organization, and nascent rRNA dynamics within a common framework, this study provides a basis for exploring the establishment, maintenance, and remodeling of distinct nucleolar profiles in cancer.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

Background. Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods. Male, 8 weeks-old mice bearing phosphatase and tensin homolog (Pten) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results. Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid {beta}-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and -linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions. Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation.

Background. Alternative oxidase (AOX) is a mitochondrial terminal oxidase that provides an alternative route for electron transfer, contributing to respiratory flexibility and redox homeostasis under stress conditions. In trypanosomatids, AOX displays a highly uneven distribution associated with extensive diversification of mitochondrial electron transport chains (ETCs). In addition to canonical AOX, many trypanosomatids encode an enigmatic AOX-like protein (AOX-L), whose evolutionary origin and functional significance remain unresolved. Results. Here, we investigated the evolutionary distribution, phylogenetic relationships, structural conservation, and functional divergence of AOX and AOX-L across euglenozoans. Comparative genomic analyses revealed a patchy distribution of both proteins among kinetoplastids, with multiple independent losses during evolution. Phylogenetic analyses demonstrated that AOX and AOX-L represent distinct evolutionary lineages, indicating independent origins rather than duplication-derived divergence. While AOX showed a conserved eukaryotic origin, AOX-L occupied a separate phylogenetic position and lacked key residues required for ubiquinol oxidation. Structural modelling revealed that AOX-L retained the characteristic AOX fold, predicted membrane association, and dimeric organization, but lacked the catalytic architecture necessary for enzymatic activity. Transcriptomic and biochemical analyses of four AOX-containing trypanosomatid species showed that AOX expression and activity correlated with mitochondrial ETC organization. Species lacking cytochrome-dependent complexes III and IV displayed substantially higher AOX expression and enzymatic activity, consistent with AOX functioning as the primary terminal oxidase in these lineages. Conversely, species retaining a canonical ETC exhibited lower AOX activity, suggesting a role in metabolic flexibility and redox regulation. Conclusions. Our study provides a comprehensive evolutionary and functional framework for alternative oxidases in trypanosomatids. We demonstrate that AOX and AOX-L are evolutionarily distinct proteins with different predicted functions: AOX maintains respiratory electron flow according to lineage-specific mitochondrial requirements, whereas AOX-L represents a structurally conserved but catalytically inactive protein family that may have acquired an alternative regulatory role. These findings highlight how mitochondrial respiratory components diversify during eukaryotic evolution and provide a basis for future investigations into the biological function of AOX-L.

Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor that plays a central role in metabolic adaptation by regulating gene expression programs involved in lipid utilization and energy homeostasis. Although the hepatic functions of PPAR and its target genes are well characterized, less is known about its transcriptional activity in extrahepatic tissues. Pemafibrate (K-877), a selective PPAR modulator (SPPARM), is a high-affinity PPAR ligand with improved potency and selectivity compared with conventional fibrates. Here, we investigated pemafibrate-induced tissue-specific transcriptional responses comparing wild type and whole-body PPAR-deficient littermate mice. Twelve-week-old male PPAR+/+ and PPAR-/- mice were treated daily with pemafibrate (0.1 mg/kg) or vehicle by oral gavage for 14 days. We performed genome-wide transcriptomic analyses in the liver, jejunum, kidney, soleus muscle, and BAT. Pemafibrate-induced transcriptional changes were entirely dependent on PPAR, as no differentially expressed genes were detected in PPAR-/- mice. The magnitude and nature of the response were tissue-specific, with the strongest transcriptional effects observed in the liver and jejunum, whereas the kidney, soleus muscle, and BAT showed limited responses. In the liver, pemafibrate primarily induced the expression of genes involved in fatty acid metabolism, mitochondrial function, and energy production. In the jejunum, pemafibrate activated lipid metabolic programs together with tissue-specific pathways related to cellular homeostasis. Collectively, our findings show that pemafibrate is a highly selective PPAR activator with a preferential transcriptional activity in the liver and intestine after oral administration. This study provides a resource for defining tissue-specific PPAR-responsive gene networks and expands our understanding of PPAR functions beyond the liver.

The phenotype of a focal individual can be affected not only by the individuals own genes (direct genetic effects, DGE) but also by genes of other individuals it interacts with (indirect genetic effects, IGE). Better understanding IGE can help better understand the impact and mechanisms of social effects. Here, we investigated, in laboratory mice, the modulation of IGE by genetic background of the focal individual (social epistasis) and by sex. We focused on IGE arising from Epha4, a gene that has been associated with the individuals own stress-related and social behaviours (i.e. DGE) and the stress-coping behaviour and wound healing of the other mice in the cage (i.e. IGE). Among four different genetic backgrounds for focal mice, significant IGE from Epha4 were detected in CBA females only. A formal model selection analysis was consistent with the modulation of IGE by the similarity of the genetic backgrounds of the focal mouse and its Epha4 cage mate, as well as by sex. Observations of fighting in cages of males with dissimilar genetic backgrounds suggested aggression might underpin these modulatory effects, which prompted to us to expand the conceptual framework describing the mechanisms of social epistasis. Thus, our results strengthen the emerging view that IGE are not fixed properties of social genotypes but can depend on the genetic and sex context of interacting individuals, revealing an underexplored layer of their genetic architecture.

The circadian clock allows living systems to anticipate environmental daily variations by scheduling cellular mechanisms according to the time of the day. The reciprocal interplay between metabolism and circadian clock is essential for energy homeostasis, and the disruption of metabolic clock inputs contributes to dysfunction of clocks in peripheral tissues. However, the molecular mechanisms through which the feeding/fasting cycle entrains peripheral clocks remain incompletely understood. Here, we investigate experimentally and theoretically the role of AMP-dependent kinase (AMPK), a key fasting sensor, in metabolic regulation of the circadian clock. Using a luciferase reporter driven by the Bmal1 promoter to monitor molecular clock activity in human U2OS cells, and thanks to a signal processing method based on the Hilbert transform to retrieve the instantaneous phase and period of circadian signals, we show that pharmacological activation of AMPK by AICAR significantly elevates Bmal1 promoter activity and markedly lengthens the clock period in a dose-dependent manner. Conversely, the clock period is shortened by the pharmacological inhibition by SR18292 of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1-alpha). Importantly, the action of AICAR is abolished by FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, demonstrating that AMPK-dependent modulation of Bmal1 promoter activity requires NAD+ availability. These experimental results are recapitulated by numerical simulations of our previously published mathematical model, where PGC-1-alpha plays a key role in mediating AMPK activity to the clock. Moreover, the comparison of experimentally measured and theoretically predicted phase response curves, following AMPK activation at different circadian phases, provides an additional validation of the model. By integrating biological experiments with mathematical modeling, our results identify a key mechanism through which metabolic factors can entrain and regulate the circadian clock via NAD+-dependent AMPK-mediated regulation of Bmal1 promoter activity.

Macaranga tanarius (Euphorbiaceae), a myrmecophilic plant grown in subtropical countries, was identified as a source of Okinawan propolis, a honeybee product, which is glandular trichomes developed on the surface of the fruits. This tissue contains a high amount of characteristic geranylated flavonoids known as nymphaeols (nymphaeol A - C and isonymphaeol B), which exhibit strong antioxidant activity. In this study, we characterized the structure of glandular trichomes of M. tanarius, showing that they are sac-like structures containing drupelet-like aggregates of secretary cells, and a subcuticular cavity was also observed. From a cDNA library, we identified two prenyltransferase (PT) genes involved in biosynthesis of nymphaeols, and a PT gene grouped in a primary metabolic PT. Our biochemical experiments demonstrated that one of the former secondary metabolic PTs (MtPT1) is a B-ring-specific geranyltransferase for eriodictyol to yield nymphaeols B and isonymphaeol B. MtPT1 was unusual for a plant PT, an enzyme that yielded multiple reaction products. The other PT (MtPT3) transferred a geranyl moiety to the A-ring of eriodictyol to form nymphaeol A. Although dimethylallyl diphosphate (DMAPP) was not recognized as its prenyl donor substrate of eriodictyol, giving a B-ring geranylated eriodyctiol as the prenyl acceptor substrate, this enzyme used DMAPP to attach the prenyl moiety to the A-ring and produced nymphaeol C. These data suggest that M. tanarius has evolved its PT function to produce a diverse range of prenylated flavonoids using a limited set of genes.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Abstract: Endocannabinoids (eCBs) like anandamide and 2-arachidonoylglycerol are endogenous lipid ligands for CB1 and CB2 G protein-coupled receptors (GPCRs). Here, we show that phosphorylation of biologically important anandamide generates naturally occurring anandamide phosphate (AEAp), which switches GPCR ligand specificity to lysophosphatidic acid (LPA) receptors (LPARs) and the primate-specific bile acid sensory receptor MRGPRX4. The kinase responsible for anandamide phosphorylation was identified asdiacylglycerol kinase (DGK) theta (DGKq or DGKQ) using activity-guided brain fractionation, inhibitor profiling, recombinant reconstitution, and DGK isozyme screening. DGKQ showed noncanonical biphasic lipid kinetics and was inhibited by phytocannabinoids, most notably tetrahydrocannabinolic acid. Thus, eCBs are not only cannabinoid receptor ligands but also enzymatically adaptable lipid signals activating LPARs and MRGPRX4, thus linking metabolism of the distinct lipid LPA with Cannabis pharmacology.

Peroxisomes, along with mitochondria, coordinate and compartmentalize oxidative metabolism in eukaryotic cells. Rare genetic disorders caused by mutations in PEX genes impair peroxisome function and cause Peroxisome Biogenesis Disorders, which are characterized by liver and neurological dysfunction, hearing and vision loss, and metabolic abnormalities. The majority of Peroxisome Biogenesis Disorders (PBDs) are caused by mutations in the gene encoding PEX1, which together with PEX6 forms a hetero-hexameric AAA-ATPase complex that drives the import of enzymes into the peroxisome lumen. One particular destabilizing mutation - PEX1G843D - results in almost 30% of all cases. Here we show that deficiencies in PEX1 lead to increased levels of the oxygen-responsive transcription factor HIF1 in normoxia, as well as a HIF1 transcriptional signature. The increase in HIF1 protein was rescued by overexpression of PEX1WT, suggesting PEX1 deficiencies modulate HIF1 signaling. The increased levels of HIF1 were not explained by defects in the oxygen responsive degradation pathway of HIF1 that relies on proline hydroxylase domain enzyme-dependent hydroxylation and von Hippel Lindau tumor suppressor protein-dependent ubiquitination. Instead, we found that PEX1 deficiency alters HIF1 proteostasis by reducing degradation through a hydroxylation- and autophagy- dependent mechanism. Notably, enhancing autophagic capacity by ULK1 agonism was sufficient to reduce HIF1 levels in PEX1 deficient cells. Lastly, we demonstrate that upon hypoxia-reoxygenation, PEX1 deficient cells are slower to reset HIF1 levels. Our results suggest that PBD patients with PEX1 deficiency may be susceptible to dysregulation of the HIF1 pathway, particularly in tissues where oxygen gradients are physiological or developmentally required.