eLife: latest articles by subject

A membrane-disruptive action of VBIT-4 challenges its role as a widely used VDAC1 oligomerization inhibitor

lbergdoll@imm.cnrs.fr (Bethel G Beyene) — Fri, 09 Oct 2026 00:00:00 +0000

Voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane and a key regulator of metabolite exchange and mitochondrial physiology. Its oligomerization has been proposed to control processes such as mitochondrial DNA release and membrane remodeling, yet the underlying mechanisms remain poorly defined. VBIT-4 has been widely used as a putative inhibitor of VDAC1 oligomerization, despite limited mechanistic validation. Here, using high-speed atomic force microscopy (AFM), we visualized VDAC1 assemblies in lipid membranes and examined the effect of VBIT-4. Unexpectedly, VBIT-4 induced membrane defects and permeabilization at micromolar concentrations, independently of VDAC1. Quantitative AFM analysis further shows that VBIT-4 does not alter VDAC1 cluster organization. Complementary electrophysiology, microscale thermophoresis, and coarse-grained molecular dynamics demonstrate that VBIT-4 partitions into lipid bilayers, increases membrane permeability, and destabilizes membrane structure, without detectable effects on VDAC1 channel properties or assemblies. Consistent with this mechanism, VBIT-4 induces VDAC1-independent cytotoxicity in HeLa cells at concentrations above 10 µM. Together, these results demonstrate that VBIT-4 does not act as a specific inhibitor of VDAC1 oligomerization but instead functions as a membrane-active compound. This work provides a revised framework for interpreting studies using VBIT-4 and highlights the importance of systematically assessing drug-membrane interactions when targeting membrane proteins.

Genome-wide synthetic lethality screen of Bam complex-associated genes in Escherichia coli

jack.bryant@nottingham.ac.uk (Charly D Neilson) — Thu, 08 Oct 2026 00:00:00 +0000

Biogenesis of the bacterial outer membrane is key to bacterial survival and antibiotic resistance. Central to this is the β-barrel assembly machine (Bam) complex and its associated chaperones, which are responsible for transport, folding, and insertion of outer membrane proteins (OMPs). The Escherichia coli Bam complex is composed of two essential subunits, BamA and BamD, and three non-essential accessory lipoproteins, BamB, BamC, and BamE. Optimal Bam function is further dependent on the non-essential periplasmic chaperones DegP, Skp, and SurA. Despite intensive study, the specific function of these non-essential Bam-associated proteins is not fully understood. Here, we analysed ΔbamB, ΔbamC, ΔbamE, ΔsurA, Δskp, and ΔdegP knockout strains by phenotypic screening, conservation analysis and high-throughput genetics. We identified hundreds of synthetic-lethal interactions and revealed that Bam complex activity is impacted by changes in outer membrane lipid composition and that enterobacterial common antigen is essential in the absence of the chaperone SurA. We also show that genes responsible for synthesis of peptidoglycan are synthetically lethal with Bam accessory lipoprotein encoding genes. Together, our data indicate potential mechanisms for coordination of OMP biogenesis with other cellular growth processes, such as LPS and peptidoglycan biogenesis.

Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation

lyonam@purdue.edu (Angeline M Lyon) — Wed, 30 Sep 2026 00:00:00 +0000

Phospholipase C β (PLCβ) enzymes are activated by heterotrimeric G protein subunits, increasing hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. All four human PLCβ isoforms (PLCβ1–4) are activated by Gα_q, whereas PLCβ1–3 are activated to varying extents by Gβγ. The binding sites for Gα_q on PLCβ are well established, and much has been learned about its mechanism of activation, but comparatively little is known about Gβγ-dependent activation. In this work, we used cryo-electron microscopy single-particle analysis, functional assays, and bioluminescence resonance energy transfer to investigate how Gβγ interacts with PLCβ3 in concert with activated Gα_q to regulate phospholipase activity. Gβγ heterodimers bind multiple surfaces of PLCβ3 to promote activation, but alone do not recruit the enzyme to the plasma membrane. Instead, Gβγ facilitates activation by Gα_q, most likely by reorienting the phospholipase catalytic site at the membrane to maximize PI(4,5)P2 hydrolysis and downstream Ca²⁺ release. Cell-based functional assays demonstrate that Gβγ is required for maximal PLCβ3 activation, even when G_q heterotrimers are the sole source of Gβγ. Together, these findings demonstrate that Gβγ acts as a critical positive allosteric modulator that regularly acts in concert with Gα_q to activate PLCβ3 at the plasma membrane.

Integrated respirometry and metabolomics unveil circadian metabolic dynamics in Drosophila

aalim@upenn.edu (Aalim Weljie) — Thu, 24 Sep 2026 00:00:00 +0000

Sleep and circadian rhythms shape organismal energy patterns, but how this timing connects to oxygen use and carbon dioxide production remains incompletely understood. We combined high-resolution respirometry with liquid chromatography-mass spectrometry (LC-MS)-based metabolomics to characterize respiratory dynamics and metabolic states in Drosophila melanogaster, resolving genotype-specific impacts of sleep disruption and circadian regulation. Wild-type flies under light-dark cycles (WT-LD) showed rhythmic respiratory patterns reflective of anticipatory coordination of mitochondrial energy metabolism, amino acid turnover, and redox cycling. Short-sleep mutants (fmn, sss) exhibited elevated metabolic rates, with reactive shifts of fuel preferences toward lipid and amino acid catabolism, and altered mitochondrial respiration. The clock mutant (per⁰¹) and flies under constant darkness (WT-DD) showed reactive and widespread metabolic dysregulation and impaired redox homeostasis. These findings demonstrate that both sleep and circadian systems contribute to aligning metabolic substrate selection with energy demands, offering mechanistic insights into how disruptions in behavioral states compromise metabolic health.

Screening the MMV Pathogen Box reveals the mitochondrial bc₁-complex as a drug target in mature Toxoplasma gondii bradyzoites

blumem@rki.de (Deborah Maus) — Thu, 17 Sep 2026 00:00:00 +0000

The apicomplexan parasite Toxoplasma gondii infects 25–30% of the global human population and can cause life-threatening diseases in immunocompromised patients. The chronically infectious forms of the parasite, bradyzoites, persist within cysts in brain and muscle tissue, and are responsible for its transmission and remission of the disease. Currently available treatment options are very limited and are only effective against the fast-replicating tachyzoites, but fail to eradicate the chronic stages of T. gondii. The cause of these treatment failures remains unclear. Here, we utilized our recently developed human myotube-based culture model to screen compounds from the MMV Pathogen Box against pan-resistant in vitro bradyzoites, and identified multiple compounds with simultaneous activity against tachyzoites and bradyzoites. Stable isotope-resolved metabolic profiling on tachyzoites and bradyzoites identified the mitochondrial bc₁-complex as a target of bradyzocidal compounds and defined their metabolic impacts on both parasite forms. Our data suggest that mature bradyzoites rely on mitochondrial ATP production.

The chromokinesin Kid (KIF22) forms a homodimer, moves processively along microtubules, and transports double-stranded DNA

shinsuke.niwa.c8@tohoku.ac.jp (Kyoko Chiba) — Wed, 16 Sep 2026 00:00:00 +0000

During prometaphase in mitosis, chromosomes are pushed toward the spindle equator. The chromokinesin Kid, also known as KIF22, moves chromosomes along spindle microtubules during prometaphase. Kid has long been considered a monomeric and nonprocessive motor, different from typical kinesins. In this study, we demonstrate that the full-length Kid forms a homodimer and moves processively along microtubules. A conserved coiled-coil domain within the stalk region of Kid is sufficient for homodimer formation and is required for the processivity of Kid. Furthermore, the neck linker and coiled-coil domains of Kid could add processive activity to the motor domain of KIF1A, suggesting that Kid contains a functional neck linker and dimerization capability, a prerequisite for the processivity of kinesin motor domains. The full-length Kid, containing a helix–hairpin–helix domain, can transport double-stranded DNA along microtubules in vitro. AlphaFold3 prediction suggests that the dimerization of Kid stabilizes the association with DNA. These findings collectively suggest the reclassification of Kid as a processive and dimeric motor that transports DNA along microtubules.

Acyl carrier protein is essential for apicoplast biogenesis in malaria parasites independent of fatty acid synthesis

sprigge2@jhu.edu (James A Wohlschlegel) — Mon, 14 Sep 2026 00:00:00 +0000

Acyl carrier protein (ACP) and its 4-phosphopantetheine prosthetic group canonically function as the soluble scaffold for acyl chain assembly and elongation during type II fatty acid biosynthesis (FASII). Plasmodium malaria parasites retain a FASII pathway in the apicoplast organelle that has been the subject of considerable scrutiny and confusion. Although apicoplast FASII is essential for Plasmodium falciparum growth within mosquitoes and the human liver, this pathway is dispensable and largely inactive in blood-stage parasites that can scavenge host fatty acids. In contrast to FASII enzymes that can be disrupted without fitness defect, we report that knockout or ligand-dependent knockdown of apicoplast ACP is lethal to blood-stage P. falciparum, indicating an essential FASII-independent function. Loss of ACP impairs the biosynthesis of essential isoprenoid precursors and blocks apicoplast biogenesis. Using proximity biotinylation and biochemical interaction studies, we identified a key role for ACP in binding and stabilizing apicoplast pyruvate kinase II (PKII). This critical enzyme is the only known source of nucleoside triphosphates (NTPs) in this organelle and is required for isoprenoid synthesis and apicoplast biogenesis. Our work reveals that ACP knockdown results in destabilization and loss of PKII, which is sufficient to explain ACP essentiality in this stage. This work unveils essential ACP function at a key biochemical hub controlling broad apicoplast metabolism in malaria parasites that is independent of the canonical ACP role in FASII.

A simplified and highly efficient cell-free protein synthesis system for prokaryotes

zhezhang@sdu.edu.cn (Changbin Zhang) — Tue, 08 Sep 2026 00:00:00 +0000

Cell-free protein synthesis (CFPS) systems are a powerful platform with immense potential in fundamental research, biotechnology, and synthetic biology. Conventional prokaryotic CFPS systems, particularly those derived from Escherichia coli, often rely on complex reaction buffers containing up to 35 components, limiting their widespread adoption and systematic optimization. Here, we present an optimized E. coli cell-free protein synthesis (eCFPS) system, which is significantly streamlined for high efficiency. Through systematic screening, we successfully reduced the essential core reaction components from 35 to a core set of 7. The thorough optimization of these seven key components ensured that protein expression levels were not only maintained but even substantially improved. Furthermore, we developed a much simpler procedure for preparing the bacterial cytosolic extracts, a ‘fast lysate’ protocol that eliminates the traditional time-consuming runoff and dialysis steps, thereby enhancing the overall accessibility and robustness of eCFPS. This optimized and user-friendly eCFPS efficiently synthesizes challenging proteins, including functional, self-assembling vimentin, and active restriction endonuclease BsaI despite its strong cytotoxicity, and serves as a powerful tool that will facilitate diverse applications in basic life science research and beyond.

Squidly harnesses enzyme functional hierarchy and contrastive learning to efficiently predict catalytic residues from sequence

amora@aithyra.ac.at (Ariane Mora) — Fri, 04 Sep 2026 00:00:00 +0000

Enzymes present a sustainable alternative to traditional chemical industries, drug synthesis, and bioremediation applications. Because catalytic residues are the key amino acids that drive enzyme function, their accurate prediction facilitates enzyme function prediction. Sequence similarity-based approaches such as BLAST are fast but require previously annotated homologues. Machine-learning (ML) approaches aim to overcome this limitation; however, current gold-standard ML-based methods require high-quality 3D structures limiting their application to large datasets. To address these challenges, we developed Squidly, a sequence-only tool that leverages contrastive representation learning with a biology-informed, rationally designed pairing scheme to distinguish catalytic from non-catalytic residues using per-token Protein Language Model embeddings. Squidly surpasses state-of-the-art ML annotation methods in catalytic residue prediction while remaining sufficiently fast to enable wide-scale screening of databases. We ensemble Squidly with BLAST to provide an efficient tool that annotates catalytic residues with high precision and recall for both in- and out-of-distribution sequences.

A whole-animal phenotypic drug screen identifies suppressors of atherogenic lipoproteins

sfarber3@jhu.edu (Adrian G Rivera Cruz) — Thu, 27 Aug 2026 00:00:00 +0000

Lipoproteins are essential for lipid transport in all bilaterians. A single Apolipoprotein B (ApoB) molecule is the inseparable structural scaffold of each ApoB-containing lipoprotein (B-lps), which are responsible for transporting lipids to peripheral tissues. The cellular mechanisms that regulate ApoB and B-lp production, secretion, transport, and degradation remain to be fully defined. In humans, elevated levels of vascular B-lps play a causative role in cardiovascular disease. Previously, we have detailed that human B-lp biology is remarkably conserved in the zebrafish using an in vivo chemiluminescent reporter of ApoB (LipoGlo) that does not disrupt ApoB function. Thus, the LipoGlo model is an ideal system for identifying novel mechanisms of ApoB modulation and, due to the ability of zebrafish to generate many progeny, is particularly amenable to large-scale phenotypic drug screening. Here, we report a screen of roughly 3000 compounds that identified 49 unique ApoB-lowering hits. Nineteen hits passed orthogonal screening criteria, and seven were subjected to extensive phenotyping. A licorice root component, enoxolone, significantly lowered B-lps only in animals that express a functional allele of the nuclear hormone receptor Hepatocyte Nuclear Factor 4⍺ (HNF4⍺). Consistent with this result, inhibitors of HNF4⍺ also reduce B-lp levels. These data demonstrate that mechanism(s) of action can be rapidly determined from a whole-animal zebrafish phenotypic screen. Given the well-documented role of HNF4⍺ in human B-lp biology, these data validate the LipoGlo screening platform for identifying small-molecule modulators of B-lps that play a critical role in a leading cause of worldwide mortality.

A tool to pulse-label yeast nuclear pore complexes in imaging and biochemical experiments

l.m.veenhoff@rug.nl (Annemiek C Veldsink) — Mon, 10 Aug 2026 00:00:00 +0000

Nuclear pore complexes (NPCs) are key gateways to the nucleus and major organizers of genome architecture. Despite their importance, it is still not fully understood how NPCs are formed and degraded. Tools to track specific NPCs over time or under stress could unlock critical insights into these questions. Here, we demonstrate that a brief pulse of expression of a previously developed nanobody against baker’s yeast nucleoporin Nup84 (Nordeen et al., 2020) enables a robust, rapid, and straightforward method for pulse-labeling NPCs in both imaging and affinity purification experiments. This approach offers an alternative to permanent, yet less rapid, genetic fluorophore- or tag-switching techniques, and provides a powerful tool for studying NPC inheritance and turnover through both microscopy and biochemical methods.

Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis

amuir@uchicago.edu (Alexander Muir) — Wed, 05 Aug 2026 00:00:00 +0000

Nutrient limitation is a characteristic feature of poorly perfused tumors. In contrast to well-perfused tissues, nutrient deficits in tumors impose metabolic constraints on cancer cells. The metabolic constraints created by the tumor microenvironment can lead to vulnerabilities in cancers. Identifying the metabolic constraints of the tumor microenvironment and the vulnerabilities that arise in cancers can provide new insight into tumor biology and identify promising anti-neoplastic targets. To identify how the microenvironment constrains the metabolism of pancreatic tumors, we challenged pancreatic cancer cells with microenvironmental nutrient levels and analyzed changes in cellular metabolism. We found that arginine limitation in pancreatic tumors perturbs saturated and monounsaturated fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1, in part via activation of the amino acid sensor GCN2. Synthesis of these fatty acids is critical for maintaining a balance of saturated, monounsaturated, and polyunsaturated fatty acids (PUFAs) in cellular membranes. Because of microenvironmental constraints on fatty acid synthesis, pancreatic cancer cells and tumors are unable to maintain lipid homeostasis when exposed to PUFAs, leading to cell death by ferroptosis. In sum, arginine restriction in the tumor microenvironment constrains lipid metabolism in pancreatic cancers, which renders these tumors vulnerable to polyunsaturated-enriched fats.

ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter

tampe@em.uni-frankfurt.de (Christoph Nocker) — Fri, 31 Jul 2026 00:00:00 +0000

ATP-binding cassette (ABC) transporters are essential molecular machines whose conformational dynamics have largely been inferred from ensemble-averaged measurements. Resolving dynamic heterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we use single-molecule Förster resonance energy transfer (smFRET) to resolve ATP-driven conformational dynamics of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human antigen transporter TAP, at the level of individual molecules. Fluorophores positioned at the nucleotide-binding domains and periplasmic gate were validated by accessible-volume simulations, fluorescence lifetimes, and ensemble FRET, demonstrating that these reporters reliably track conformational transitions. Single-molecule analysis distinguishes ATP-free and ATP-bound states and quantifies ATP-dependent population shifts from nucleotide-free to physiological ATP concentrations. Kinetic analysis further reveals an unexpectedly long ATP-bound dwell time of ~300 ms. Using complementary stabilization strategies, we directly resolve a previously hidden outward-facing open state that is kinetically masked under turnover conditions. These results provide the first single-molecule characterization of TmrAB and establish a quantitative single-molecule framework for dissecting ATP-coupled conformational dynamics in heterodimeric ABC transporters.

Repurposed small molecule toxin inhibitors neutralise a diversity of venoms from the Neotropical viperid snake genus Bothrops

rachel.clare@edgehill.ac.uk (Adam Westhorpe) — Fri, 31 Jul 2026 00:00:00 +0000

Snakebite globally claims more than 100,000 lives per year and results in morbidity for 400,000 survivors. Current treatment uses antibody-based antivenoms which are constrained by their efficacy, safety, and cost. In this study we evaluated the efficacy of previously described repurposed drugs against viperid snakes of the medically important Bothrops genus. Despite variable toxin representation and bioactivity across this central and south American genus, we found that the lead inhibitors targeting metalloproteinases (marimastat and DMPS) and phospholipases (varespladib) demonstrated pan-species neutralisation in enzymatic assays, whilst nafamostat (serine protease inhibitor) had variable activity. The metalloproteinase inhibitors protected against the procoagulant and haemorrhagic effects of several venoms in phenotypic assays. Collectively these findings demonstrate that repurposed drugs may be of great value as early interventions for the treatment of bothropic envenoming in the Neotropics and thus provide a strong rationale for their progression into future preclinical and clinical evaluation for snakebite indication.

DNA tensiometer reveals catch-bond detachment kinetics of kinesin-1, -2, and -3

woh1@psu.edu (Crystal R Noell) — Mon, 20 Jul 2026 00:00:00 +0000

Bidirectional cargo transport by kinesin and dynein is essential for cell viability, and defects are linked to neurodegenerative disease. Computational models predict that load-dependent motor detachment strongly determines the outcome of kinesin–dynein tug-of-war, with kinesin-3 and kinesin-2 more load-sensitive than kinesin-1. Yet reconstituted assays show that all three kinesin families compete similarly well against dynein. Previous work demonstrated that vertical forces from optical trapping assays can enhance kinesin-1 dissociation, suggesting that motor behavior may depend strongly on cargo geometry. To measure kinesin detachment and reattachment kinetics under forces applied parallel to the microtubule, we developed a DNA-based tensiometer using an entropic DNA spring linking motors to microtubules. For kinesin-1, –2, and –3, dissociation rates at stall were slower than during unloaded motion, and reattachment kinetics were consistent with a weakly bound slip state preceding detachment. Kinesin-3 behavior further suggested that long KIF1A run lengths arise from multiple short runs connected by diffusive episodes. Stochastic simulations reproduced the measured load-dependent kinetics and enabled direct comparison of transition rates among kinesin families. These results provide insight into how kinesin-1, –2, and –3 transport cargo in complex cellular geometries and compete against dynein during bidirectional transport.

Experimental verification of the error minimization theory using non-standard genetic codes constructed in vitro

ichihashi@bio.c.u-tokyo.ac.jp (Norikazu Ichihashi) — Mon, 13 Jul 2026 00:00:00 +0000

All living systems use an almost identical standard genetic code (SGC), in which 20 amino acids are assigned non-randomly. According to the error minimization theory, amino acids are arranged to minimize the mutational effect on protein function, while experimental verification remains limited. Here, we constructed 10 non-standard genetic codes (non-SGCs) in vitro by reassigning three amino acids (Ala, Ser, and Leu) in vacant codons of the minimal genetic code consisting of 21 tRNAs. Most of these non-SGCs have a higher cost of amino acid replacement than the SGC, calculated based on three amino acid properties: polar requirement (PR), molecular volume (MV), and hydropathy index (HI). The protein function of three reporter genes expressed using these non-SGCs decreased similarly when random mutations were introduced into the genes, implying that the effect of mutations was similar across all the non-SGCs tested here. This result provides direct experimental evidence that mutational robustness does not significantly change in individual reporter protein activity within the range of mutational cost tested in this study (Cost_PR: 5.29–5.77, Cost_MV: 1848–2348, and Cost_HI: 3.27–5.10), which covers approximately 18.4% (PR), 37.6% (MV), and 50.8% (HI) of the possible cost range achievable among one million randomly-generated genetic codes.

Dynamic assembly of malate dehydrogenase–citrate synthase multienzyme complex in the mitochondria

tobata2@unl.edu (Connor Pedersen) — Wed, 08 Jul 2026 00:00:00 +0000

The tricarboxylic acid (TCA) cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) form a multienzyme complex, referred to as a metabolon, that channels intermediate oxaloacetate between their reaction centers. Given that the MDH1–CIT1 metabolon enhances pathway reactions in vitro, its dynamic assembly is hypothesized to contribute to TCA cycle regulation in response to cellular metabolic demands. Here, we demonstrated that yeast mitochondrial MDH1 and CIT1 dissociated when aerobic respiration was suppressed by the Crabtree effect and associated when the respiratory activity was enhanced by acetate. Pharmacological TCA cycle inhibition dissociated the complex, whereas electron transport chain inhibition enhanced the interaction. The multienzyme complex assembly was related to the mitochondrial matrix acidification and oxidation, as well as cellular levels of malate, fumarate, and citrate. These factors significantly affected the MDH1–CIT1 complex affinity in vitro. Especially, variations in buffer pH within the physiological pH range between 6.0 and 7.0 in the mitochondrial matrix significantly impacted the MDH1–CIT1 affinity. These results demonstrate the dynamic association and dissociation of the MDH1–CIT1 metabolon and its relationship with respiratory activity, supporting metabolon dynamics as an integral factor in metabolic regulation governed by multiple factors such as mitochondrial pH and metabolite levels.

Profiling of terminating ribosomes reveals translational control at stop codons

sq38@cornell.edu (Leiming Dong) — Tue, 07 Jul 2026 00:00:00 +0000

Accurate termination of protein synthesis is paramount for the integrity of the cellular proteome, yet the dynamics and fidelity of ribosome termination remain poorly understood. Here, we establish a profiling strategy to capture terminating ribosomes in mammalian cells and reveal a substantial heterogeneity in ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that promotes termination pausing, a finding supported by massively parallel reporter assays. Unexpectedly, reduced termination pausing increases the likelihood of stop codon slippage, giving rise to proteins with heterogeneous C-terminal extensions. Mechanistically, we show that sequence-dependent termination pausing is consistent with post-decoding mRNA scanning by the 3′ end of 18 S rRNA. We further uncover tissue-specific patterns of termination pausing that correlate with the stoichiometry of Rps26, which potentially modulates mRNA:rRNA interactions. Together, these results suggest termination pausing as a distinct translational signature shaped by mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.

Non-canonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

jiouw@jhu.edu (Hao Chen) — Fri, 03 Jul 2026 00:00:00 +0000

We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent non-canonical amino acid Anap. By integrating the genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags, and enabling physiologically relevant visualization of protein pathobiology.

Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry

Sonia.Rocha@liverpool.ac.uk (Angus I Lamond) — Thu, 25 Jun 2026 00:00:00 +0000

We have developed a robust workflow to identify proline hydroxylation sites in proteins, combining hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-liquid chromatography-mass spectrometry (LC-MS) with refining and filtering parameters during data analysis. Using this approach, we have combined data from cell lines treated with either the prolyl hydroxylase (PHD) inhibitor, Roxadustat (FG-4592), or with the proteasome inhibitor MG-132, or with a DMSO control, to identify a total of 4993 and 3247 proline hydroxylation sites, respectively, in HEK293 and RCC4 cells. Of these, 1954 (HEK293) and 1253 (RCC4) high-confidence non-collagen sites were inhibited by FG-4592. Hydroxylated peptides showed consistent characteristics across both datasets, including enrichment in more hydrophilic HILIC fractions and distinct charge and mass distributions compared to unmodified or oxidised peptides. The intensity of the diagnostic hydroxyproline immonium ion varied with MS collision energy, peptide concentration, and adjacent amino acid sequence. Using synthetic peptides, we demonstrate that combining LC retention time with optimised MS parameters enables reliable site identification, even with multiple proline residues present. Proteins with FG-4592-inhibited hydroxylation sites were enriched for roles in RNA metabolism, mRNA splicing, and cell cycle regulation, including the phosphatase 1 regulatory subunit Repo-Man (CDCA2).

Structural insights into the recruitment of viral type 2 IRES to ribosomal preinitiation complex for protein synthesis

hussain@iisc.ac.in (Deepakash Das) — Thu, 25 Jun 2026 00:00:00 +0000

Picornaviruses employ internal ribosome entry sites (IRESs) in their genomic RNA to hijack the host’s translational machinery. The picornavirus, encephalomyocarditis virus, employs a type 2 IRES present in its 5’ untranslated region (5’UTR) and requires 43S ribosomal preinitiation complex (PIC), the central domain of eukaryotic initiation factor (eIF) 4G, eIF4A, and an essential ITAF (IRES trans-acting factor)-polypyrimidine tract binding protein 1 (PTB1) to form 48S PIC. In this study, we have used cryo-electron microscopy (cryo-EM) to determine the structure of encephalomyocarditis virus (EMCV) IRES-bound mammalian 48S PIC in a scanning-arrested closed state at the start codon. The EMCV IRES domains contact initiator tRNA (tRNA_i) and 40S head at the inter-subunit interface, which reveals an altogether unique mechanism used by viruses to capture host translational machinery for its protein synthesis. The tRNA_i is held away from the 40S body in contrast to canonical cap-dependent translation while the domain I apical region of EMCV IRES mimics 28S rRNA of 60S to interact with 40S ribosomal head proteins uS13 and uS19. The structural analysis accounts for numerous previously reported biochemical studies on type 2 IRES and shows how type 2 IRES interacts with 43S PIC to form 48S PIC. This study provides mechanistic insights for understanding EMCV IRES-mediated translation initiation, which could be extrapolated to other IRESs sharing similar motifs and factor requirements, including type 1 viral IRESs.

Correction: SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

Wed, 24 Jun 2026 00:00:00 +0000

Exploration of precision coregulator TR-FRET identifies diverse signatures for LXR ligands relevant to discovery of nonlipogenic ABCA1 inducers

grjthatcher@arizona.edu (Anandhan Annadurai) — Mon, 22 Jun 2026 00:00:00 +0000

APOE4, the major genetic risk factor for Alzheimer’s disease (AD), and ATP-binding cassette-A1 (ABCA1), required for lipidation of APOE are gene products of the liver X receptor (LXR) receptor. LXR agonists have been validated in animal models as therapeutics for AD, atherosclerosis, and many other diseases. Clinical progress has been thwarted by unwanted hepatic lipogenesis. Structurally diverse LXR ligands were profiled in coregulator TR-FRET (CRT) assays analyzing ligand-induced coactivator recruitment, coactivator selectivity, corepressor dissociation, and LXR isoform selectivity. A multiplex CRT assay was developed to measure synchronous ligand-induced displacement of corepressor by coactivator. Potency for coactivator recruitment to LXRβ correlated with induction of ABCA1 in human astrocytoma cells. Correlation with lipogenic activation of sterol response element (SRE) in hepatocarcinoma cells, was more complex. CRT response was diverse revealing ligands with theoretical full agonist, partial agonist, antagonist, inverse agonist, and other signatures within the same chemical series, suggesting the scope for precision CRT to guide nonlipogenic LXR agonist design.

Single-step in vitro reconstitution of the Escherichia coli ribosome mediated by two GTPase factors, EngA and ObgE

yshimizu@riken.jp (Aya Sato) — Thu, 18 Jun 2026 00:00:00 +0000

When Escherichia coli ribosomes are assembled in vitro, manipulation of incubation temperature and magnesium ion concentration has been an essential procedure, which is a crucial step for the assembly of active large subunits. The present study tackles this issue to develop a single-step procedure, which can be performed in near-physiological conditions, where cell-free protein synthesis is active. We found that GTPase factors EngA and ObgE can complement the changes in temperature and magnesium ion concentrations. In the presence of these factors, both the ribosome assembly and translation processes were successfully integrated in the reconstituted cell-free protein synthesis system. Furthermore, we found that these GTPase factors can reassemble the ribosomes to an active state, whose structure was disrupted by EDTA chelation of magnesium ions, indicating that these two factors can reversibly induce the ribosome structure to an intact state. The findings are essential for the bottom-up construction of synthetic cells.

Nuclear CK1δ as a critical determinant of PER:CRY complex dynamics and circadian period

michael.brunner@bzh.uni-heidelberg.de (Axel CR Diernfellner) — Mon, 15 Jun 2026 00:00:00 +0000

The mammalian circadian clock is governed by a feedback loop in which the transcription activator CLOCK:BMAL1 induces expression of its inhibitors, PERs and CRYs, which form a complex with CK1δ, the main circadian kinase. However, the spatiotemporal dynamics of this feedback loop and the precise role of CK1δ remain incompletely understood. Using an inducible overexpression system, we show that nuclear availability of CK1δ is limited by both rapid nuclear degradation and active export of unassembled kinase, while cytoplasmic kinase is readily available for association with PERs. We demonstrate that CK1δ-mediated phosphorylation may disrupt PER2–CRY1 interaction, thereby resulting in cytoplasmic PER2 dimers containing substoichiometric amounts of CRY1. Analysis of endogenous PER2 localization in the context of an intact circadian clock reveals that PER2 accumulates in the cytoplasm late in the circadian cycle. Based on these findings, we propose that cytoplasmic accumulation of PER:CRY:CK1δ complexes contributes to the clearance of nuclear PER2, while the CK1δ-dependent release of CRY1 into the nucleus may sustain CLOCK:BMAL1 repression on DNA, supporting the transition from the early to the late repressive phase.

Cell size modulates ferroptosis susceptibility

ez225@cam.ac.uk (Evgeny Zatulovskiy) — Wed, 10 Jun 2026 00:00:00 +0000

Size is a fundamental property of cells that influences many aspects of their physiology. This is because cell size sets the scale for all subcellular components and drives changes in the composition of the proteome. Given that large and small cells differ in their biochemical composition, we hypothesized that they should also differ in how they respond to signals and make decisions. Here, we investigated how cell size affects the susceptibility of human cells to cell death. We found that large cells are more resistant to ferroptosis caused by system x_c^- inhibition. Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of toxic lipid peroxides. This process is opposed by cysteine-dependent lipid peroxide detoxification mechanisms. We found that larger cells exhibit higher concentrations of the cysteine-containing metabolite glutathione and lower concentrations of membrane lipid peroxides. Mechanistically, this can be explained by the fact that larger cells had lower concentrations of an enzyme that enriches cellular membranes with peroxidation-prone polyunsaturated fatty acids, ACSL4, and increased concentrations of the glutathione-producing enzymes glutamate-cysteine ligase and glutathione synthetase, the iron-chelating protein ferritin, and the lysosomal protease cathepsin B, which can catabolize cysteine-rich extracellular proteins to produce additional cystine for fueling the synthesis of glutathione. Taken together, our results highlight the significant impact of cell size on cellular function and survival, revealing a size-dependent vulnerability to ferroptosis that could influence therapeutic strategies based on this cell death pathway.

Purified zymogens reveal mechanisms of snake venom metalloproteinase auto-activation

imre.berger@bristol.ac.uk (Alastair Poole) — Wed, 10 Jun 2026 00:00:00 +0000

Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans, they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain an SVMP cocktail and due to their cytotoxicity, SVMP characterisations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII, and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn²⁺ ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterised using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology and discovery of novel snakebite therapeutics.

Effects of residue substitutions on the cellular abundance of proteins

lindorff@bio.ku.dk (Kresten Lindorff-Larsen) — Thu, 28 May 2026 00:00:00 +0000

Multiplexed assays of variant effects (MAVEs) make it possible to measure the functional impact of all possible single amino acid residue substitutions in a protein in a single experiment. Combination of variant effect data from several such experiments provides the opportunity to conduct large-scale analyses of variant effect scores measured across proteins, but can be complicated by variations in the phenotypes that are probed across experiments. Thus, using variant effect datasets obtained with similar MAVE techniques can help reveal general rules governing the effects of amino acid variation for a single molecular phenotype. In this work, we accordingly combined data from six individual variant abundance by massively parallel sequencing (VAMP-seq) experiments and analysed a total of 31,614 variant effect scores reporting solely on the impact of single amino acid residue substitutions on the cellular abundance of proteins. Using our combined variant effect dataset, we derived and analysed a collection of amino acid substitution matrices describing the average impact on cellular abundance of all residue substitution types in different structural environments. We found that the substitution matrices predict the cellular abundance of protein variants with surprisingly high accuracy when given structural information only in the form of whether a residue is buried or exposed. We thus propose our substitution matrix-based predictions as strong baselines for future abundance model development.