The FEBS Journal

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

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.

Microbial modular rhodopsins, in which light-sensing rhodopsin domains are fused with effector modules, have emerged as promising tools for optogenetic regulation in algae and other systems. However, the diversity and potential regulatory roles of fungal modular rhodopsins remain largely unexplored. Here, we performed a comprehensive in-silico analysis to identify previously uncharacterized fungal modular-rhodopsins that pair a conserved light-sensing core with diverse effector domains, including RPEL-motif, NADP-binding Rossmann fold domain, MCM (Mini-Chromosome Maintenance) domain, and GC-cAT (Carnitine O-Acetyltransferase) modules. In Aureobasidium pullulans, the representative modular rhodopsin (ApRh-RPEL) contains RPEL-motif associated with actin-related and transcriptional regulatory processes, suggesting light-driven fungal signaling pathway involved in transcriptional and cellular regulation, respectively. Rhodopsins fused with NADP-binding Rossmann fold and MCM domains further indicate possible applications in light-programmable metabolic and cell-cycle signaling. Genome mining additionally revealed that A. pullulans harbours a diverse but underexplored array of biosynthetic gene clusters (BGCs), raising the intriguing possibility that light perception may regulate secondary metabolite pathways. Supporting this, multisource protein-protein interaction network analysis links ApRh-RPEL to enzymes involved in terpenoid and sphingolipid biosynthesis, indicating potential cross-talk between light-sensing module and metabolic regulation. These findings outline a computationally derived model in which fungal modular rhodopsins (ApRh-RPEL) function as opto-synthetic regulators of biosynthetic processes. Structural predictions confirmed conserved Schiff-base lysine and retinal-binding pocket, highlighting functional diversity across fungal rhodopsins. Together, these findings expand the optogenetic toolkit and provide a framework for engineering light-driven signaling in fungi, with applications in optobiological and biomedical applications.

Calpains constitute an ancient, extensive family of calcium-dependent cysteine proteases found in some bacteria and most eukaryotes. They are involved in a wide variety of developmental and cellular processes and are implicated in major human diseases, yet it remains to be seen if they have a common core function explaining their widespread and varied presence across taxa. Beyond their core CysPc catalytic domain, calpains contain diverse domain combinations and can be either cytosolic or membrane bound. Here we hypothesize a general role for both cytosolic and transmembrane calpains in cellular cytokinesis through positional anchoring and organization of microtubules (MTs). We propose that during plant cell division, the singular transmembrane calpain DEK1 localizes and organizes the array of cortical MTs from the microtubule organizing center (MTOC) to establish the location of the preprophase band and/or the site of cell plate formation according to the positional activation of DEK1 proteins in the nuclear membrane. Similarly, during cell division in animals, their calpains may be involved in setting the point of membrane invagination via their association with membrane-bound proteins. This proposition adds to the current picture of animal MTOC/centrosome function and suggests how a calcium peak during the initial cytokinetic furrowing might be transmitted. We discuss this novel mechanistic model for calpain activity in the context of data from the animal and plant literature, as well as of our novel discovery here of calpain sequences in both brown and red algal genomes. Finally, we speculate that the ancestral role of calpains in early eukaryotes, before the split into the major eukaryotic supergroups, may have been to facilitate the formation and function of MT arrays in flagella and cilia. From this origin, calpains may have developed new functions in eukaryote cell division processes by anchoring centrosomes/MTOC to set the cell division orientations that are especially important for complex multicellularity.

A hexameric clock protein KaiC exhibits a 24-hour phosphorylation cycle with a unique property termed temperature compensation. The period is kept constant over physiological temperatures through compensatory coordination of underlying elementary reactions. The temperature-compensated ATPase activity of KaiC is one such key reactions that potentially contribute to maintaining a constant circadian period. We identified four amino acid residues responsible for the temperature compensation in an N-terminal ATPase domain of KaiC. D82 and K172 were located in a primary site, and the ATPase activity of each alanine mutant showed a positive correlation with rising temperature. N62 and E69 constituted a secondary site, where each alanine replacement resulted in a negative correlation with the temperature. The primary site exerts a compensatory regulation over the ATPase cycle locally within the N-terminal domain. The secondary site prevents the ATPase activity from becoming over-compensated by suppressing another compensatory regulation mediated through a non-local interaction with a C-terminal domain of KaiC. Therefore, any imbalance between the local and non-local compensatory regulations in KaiC affects the temperature dependence of its phosphorylation rhythm.

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

The human mitochondrial amidoxime reducing component 1 (mARC1) is a molybdenum-dependent enzyme whose protein-coding variants confer protection against common metabolic liver diseases. Whereas the frequent A165T variant acts largely through accelerated cellular degradation, the basis of protection by the rarer M187K variant remains obscure, as it has been reported that there are no differences between the "wild-type" and M187K variant protein in terms of cellular protein levels and localisation. Here, the crystal structure of the human mARC1 M187K variant, crystallised as a T4 lysozyme fusion after iterative micro-seeding, was determined at 1.63 [A] resolution. The variant structure is essentially superimposable with the previously reported "wild-type" and A165T structures, with pairwise root-mean-square deviations of 0.3-0.4 [A], and the pentacoordinated molybdenum cofactor is fully intact. Differential scanning fluorimetry across a broad pH range revealed only a modest, pH-dependent decrease in thermal stability associated with the exchange, most pronounced at alkaline pH. These data suggest that the disease-protective effect of M187K is unlikely to originate from gross structural rearrangement or active-site perturbation. SynopsisThe structure of the disease-relevant human mARC1 M187K variant was determined at 1.63 [A] resolution after iterative micro-seeding. The variant does not display relevant perturbations of the overall protein fold or active site structure, but differential scanning fluorimetry detects a pH-dependent decrease in overall protein stability associated with the M187K amino acid exchange.

Trimethylation of lysine 4 of histone H3 (H3K4me3) is a post-translational modification (PTM) enriched at promoters of actively transcribed genes. H3K4me3 is removed by the human histone demethylases of the KDM5 family. KDM5 demethylases act as transcriptional repressors through their catalytic activity in addition to more complex roles that depend on their interactions with other chromatin regulators and may be independent of demethylase activity. To better understand the mechanistic differences of the closely related paralogs KDM5A and KDM5B as well as their interactions with Retinoblastoma protein (RB), we systematically analyzed and compared their demethylase activities, nucleosome engagement, and RB binding. We used recombinant nucleosome binding and demethylase activity assays, as well as an integrative structural biology approach using negative-stain electron microscopy (EM), AlphaFold predictions, and cross-linking mass spectrometry for a comprehensive in vitro analysis of these critical and largely non-redundant enzymes. KDM5A and KDM5B showed differences in enzyme kinetics using peptide substrates, as well as in nucleosome binding. Furthermore, KDM5A interacts with RB, mainly mediated by its canonical LxCxE RB binding motif. KDM5B, on the other hand, lacks an LxCxE binding motif and does not stably bind to RB under the conditions tested here. RB directly interacts with nucleosomes, and its nucleosome binding does not measurably affect KDM5A demethylase activity or nucleosome interactions. Our findings provide a biochemical framework for the differences between KDM5A and KDM5B regarding RB interactions and nucleosome engagement.

Angiotensin-converting enzyme 2 (ACE2) functions as the receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the cellular endocytic machinery for entry by binding to defined residues on ACE2 with its spike protein (S protein), whose activation requires a priming process by another transmembrane protease, the transmembrane protease serine 2 (TMPRSS2). In addition, ACE2 itself is cleaved by TMPRSS2, which has been shown to be critical for viral pathology. This study aimed to elucidate the relationship between ACE2 and TMPRSS2 and the mechanism of ACE2 processing under normal cellular conditions. It is shown that interaction of ACE2 with TMPRSS2 results in altered processing, modification and cellular localization. Glycosylation of ACE2 has a major impact on TMPRSS2 interaction, trafficking and shedding of the enzyme. Studies in newly generated TMPRSS2-knockout rats reveal increased ACE2 levels in tissues supporting an important role of TMPRSS2 in ACE2 shedding also in vivo.

Protein homeostasis - i.e., proteostasis - is the biological process by which the qualitative and quantitative balance of the proteome is conducted, either by preserving functionally relevant proteins or by degrading unnecessary ones. Stress conditions can modulate cellular proteostasis in order to promote cytoprotection and preserve the viability of living organisms. Among the cellular pathways already described that can play an important role in preserving biological functions by modulating proteostasis are the heat shock response and the ribosome quality control pathways. In this work, we show that the Rqc1p protein is necessary for the thermoadaptation of S. cerevisiae to heat shock, as RQC1-deficient yeast is sensitive to elevated temperatures. In silico approaches - such as multiple sequence alignment, structural analysis, and molecular dynamics simulations - confirmed earlier predictions that Rqc1p shares characteristics with the bHLH family of proteins. We also verified, through computational prediction of sub-cellular localization, that S. cerevisiae Rqc1p contains nuclear localization signals, suggesting that this protein can potentially be translocated toward the nucleus, thereby broadening its current range of recognized biological functions in this organism. Also, analysis of yeast transcriptomes subjected to heat shock showed that Rqc1p mRNA levels do not fluctuate in response to heat shock, suggesting that cellular concentrations of Rqc1p are already at optimal levels to elicit a rapid and effective response during thermal stress in S. cerevisiae.

-Lipoic acid (LA) is widely included in "mitochondrial cocktails" recommended to patients with primary mitochondrial disorders, yet its mechanism of action remains unclear. Here, we define the intracellular availability and functional utilization of LA in mammalian cells. We show that LA exists in two functionally distinct cellular pools: a low-abundance free pool and a protein-bound pool generated through mitochondrial fatty acid synthesis (mtFAS). Disruption of the mtFAS pathway abolishes protein lipoylation and impairs oxidative phosphorylation without altering free LA levels. Conversely, supplementation with exogenous LA markedly increases free intracellular LA without restoring protein lipoylation, mitochondrial respiration, or cell proliferation. Instead, the cellular effects of LA supplementation resemble those of the antioxidant N-acetylcysteine. These findings clarify the mechanism of action of a widely used mitochondrial supplement and identify a fundamental disconnect between cellular LA abundance and mitochondrial utilization, challenging the rationale for using LA supplementation to restore mitochondrial function.

Old Yellow Enzymes (OYEs) are a widely distributed family of ene-reductases that were first described in a Saccharomyces cerevisiae ferment. In plants, cis-12-oxo-phytodienoic acid (cis-OPDA) reductase (OPR) is the best studied OYE. In Arabidopsis thaliana, the peroxisomal AtOPR3 was characterized as the major OPDA reductase, which generates 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid in the jasmonic acid (JA) biosynthesis. In Atopr3 lines, only small amounts of JA are detectable after wounding. Here, we describe an OPR-like enzyme (named BnOPR) from the gram-positive Brevibacillus nitrificans. The sequence was identified in an early version of the Physcomitrium patens genome and is assumed to be a contamination by a bacterium growing in association with P. patens. In complementation experiments with an Atopr3 line, we demonstrate that expression of BnOPR, fused with a peroxisomal targeting signal, rescues the male infertile phenotype and increases JA and JA-Ile levels. The catalytic parameters of BnOPR were determined for a set of substrates, including cis-OPDA and prednisone. Interestingly, B. nitrificans, B. brevis, and Paenibacillus physcomitrellae were shown to have a positive effect on P. patens growth. HighlightThe bacterial enzyme BnOPR rescues the male infertile phenotype of Atopr3 plants.

AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AfG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/720153v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4b9b5org.highwire.dtl.DTLVardef@1cc2242org.highwire.dtl.DTLVardef@dd211borg.highwire.dtl.DTLVardef@1a87722_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFtsH-IMS forms a homo-dimer in solution, whereas paraplegin-IMS presents as a well-folded monomer in solution C_LIO_LIparaplegin-IMS crystallises as a domain-swapped homo-dimer but its domain-swapped monomers are structurally similar to other IMS-regions C_LIO_LIAfG3L2/paraplegin hexamer formation may be supported by domain swapping in paraplegin-IMS C_LIO_LIdomain-swapping in paraplegin could be a Bonafide feature under certain cellular conditions and may be related to disease in spastic paraplegia C_LI

The chemokine-like peptide GPR15LG is a known agonist of G protein-coupled receptor 15 (GPR15), a ligand-receptor pair primarily implicated in mammalian mucosal immunity and lymphocyte homing. However, the evolutionary origin and phylogenetic distribution of this signaling system remain poorly understood due to the extreme sequence diversity of GPR15LG orthologs. In this study, we identified GPR15LG orthologs in several fish species for the first time according to their conserved gene synteny, genomic organization, and amino acid sequence features. A representative ortholog from the spotted gar (Lepisosteus oculatus), termed Lo-GPR15LG, was recombinantly prepared and functionally characterized using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and homogenous ligand-receptor binding assay. Our results demonstrated that Lo-GPR15LG directly binds to and efficiently activates its cognate receptor, Lo-GPR15, with a dissociation constant (Kd) of approximately 60 nM and an EC50 value of approximately 10 nM. Functional assays further revealed that receptor activation is critically dependent on the conserved C-terminal residues. Notably, human and fish orthologs exhibited no cross-species activity, consistent with their high sequence divergence. These findings reveal that the GPR15LG-GPR15 signaling system originated in ancient fish ancestors and has remained a conserved signaling axis throughout vertebrate evolution, suggesting a fundamental role in immunity across all vertebrate lineages.

The yeast secreted proteome plays critical biological roles and influences product and production parameters in industrial fermentation. Systematic profiling of the response of the yeast secretome to intrinsic and extrinsic factors is therefore essential for understanding these functions and for optimizing manufacturing processes. Here, we characterized the yeast secretome under diverse proteosynthetic stress conditions, including glycosylation deficiency, oxidative, reductive, and thermal stresses. The secretome was predominantly composed of conventionally secreted proteins, while a subset of proteins appeared to be secreted via unconventional pathways. Distinct secretome profiles were observed in response to different stressors, driven by a combination of altered intracellular proteomes, altered canonical secretion, and altered cell lysis and unconventional protein secretion, while reflecting the underlying metabolic state of the cells. Heat stress did not impact protein glycosylation but did cause similar protein misfolding stress to N-glycosylation deficiency. Intriguingly, canonically intracellular chaperone BiP was abundant in the secretome in particular stress conditions where its activity would be beneficial. BiP interacted with probable extracellular client proteins in vitro, consistent with it acting as a functional extracellular chaperone/holdase in conditions such as reductive stress in which client proteins could be misfolded outside the cell.

Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe's effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAHenu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation.

Liver sinusoidal endothelial cells (LSECs) separate the blood from the hepatic parenchyma and thus are at the frontline as scavengers of blood-borne waste, pathogens and metabolic stimuli. LSECs are also critical for sensing systemic iron availability by controlling the synthesis of bone morphogenetic protein (BMP) 6, which is essential for hepcidin expression in hepatocytes. Hepcidin maintains systemic iron homeostasis by inhibiting dietary iron uptake and iron release from iron recycling macrophages. Hepcidin is also an acute-phase protein and its activation by inflammation requires active BMP signaling. It is incompletely understood how signals derived from inflammation, cellular damage and iron are integrated by the liver to assure adequate hepcidin expression. Here, we show that Bmp6 expression is activated in primary LSEC cultures upon their exposure to danger-associated molecular patterns (DAMPs), such as heme and myoglobin, pathogen-associated molecular pattern (PAMPs), such as lipopolysaccharide (LPS) and Fibroblast-Stimulating Lipopeptide-1 (FSL1), or oxidative stress inducers (H2O2). Interestingly, all regulatory cues converge at the MAPK signaling pathway, although the specific signaling branches involved are stimulus-specific. Of note, Bmp6 upregulation in LSECs in response to all signals tested is strongly enhanced by the hepatocyte secretome. As hepatocytes critically depend on active BMP/SMAD signaling to control hepcidin activation, our results reveal that multiple sources of signaling input activating Bmp6 in LSECs and hepcidin in hepatocytes serve to determine BMP/SMAD signaling strength. Furthermore, our findings identify hypoferremia (low plasma iron levels), the result of high hepcidin levels due to elevated Bmp6, as a convergent response in conditions of inflammation, oxidative stress and cellular damage. HighlightsO_LIDAMPs (heme and myoglobin), PAMPs (LPS) and oxidative stress activate Bmp6 mRNA expression via the MAPK signaling pathway C_LIO_LIThe TLR/MAPK/BMP6 regulatory axis integrates inflammatory and iron signals C_LIO_LIOur work uncovers a novel connection between innate immune sensing, oxidative stress and hepatic iron homeostasis C_LI

Dietary methionine restriction has been shown to improve metabolic health and treat multiple diseases. Methionine metabolism regulates transmethylation reactions, including N6-methyladenosine (m6A) RNA methylation, by modulating the availability of S-adenosyl methionine (SAM). Both m6A RNA methylation and methionine metabolism are involved in the regulation of the circadian clock. However, it remains unclear whether dietary methionine influences circadian rhythms through the regulation of m6A RNA modification. In this study, we investigated the effects of short-term methionine deprivation on the diurnal oscillations of m6A RNA methylation in the mouse liver. We found that a methionine-deficient (MD) diet reprogrammed the cyclic expression patterns of m6A writers, erasers, and readers. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that the MD diet induced de novo diurnal m6A oscillations in genes associated with RNA processing, protein translation, protein ubiquitination, and mTORC1 signaling pathways. RNA-seq and quantitative proteomics analyses demonstrated that MD-induced changes in m6A RNA levels were linked to alterations in mRNA and protein abundance. We observed that dynamic m6A RNA methylation of the transcripts encoding two key enzymes, MAT2A and CBS, helps maintain methionine homeostasis in response to methionine starvation. These findings identify m6A RNA methylation as a key mechanism linking methionine metabolism to circadian regulation.

Nearly all bacteria are surrounded by a mesh-like macromolecule called peptidoglycan that gives them their shape and helps them resist turgor pressure. To grow and maintain their peptidoglycan, bacteria produce a wide range of enzymes, including the relatively understudied ,[x1D05]-transpeptidase (LDT) family. LDTs can catalyse several different reactions and vary widely in copy number: some bacteria have none, whilst others have more than twenty. To better understand why some bacteria have so many LDTs, we examined 18 putative ones from Rhizobium johnstonii, a nitrogen-fixing, symbiotic bacterium. Heterologous expression revealed several highly active enzymes, one of which, LdtRj8, we further characterized in detail. In vitro assays showed that LdtRj8 was capable of ,[x1D05]-transpeptidation, carboxypeptidation, substitution, and endopeptidation, but that its preferred activity differed at different pHs. LdtRj8 particularly excelled at ,[x1D05]-substitution, utilizing all of the tested [x1D05]-amino acids, and, surprisingly, most of the -amino acids as well. LdtRj8's pH-modulated activity could help R. johnstonii respond to acidic conditions encountered throughout the rhizobium-legume symbiosis, and its -amino acid substitution activity, which we show to be a more general property of LDTs, may regulate ,[x1D05]-transpeptidation and explain the existence of isomeric muropeptides often reported in the literature.