bims-proteo 2025-06-01 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–06–01
forty-four papers selected by
Eric Chevet, INSERM

Endoplasmic reticulum tubule junctions are sites of autophagy.
TRIP12 structures reveal HECT E3 formation of K29 linkages and branched ubiquitin chains.
Insights into the role of collided ribosomes during the activation of the integrated stress response.
The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.
Human CCR4 deadenylase homolog Angel1 is a Non-Stop mRNA Decay factor.
EndoMAP.v1 charts the structural landscape of human early endosome complexes.
Endolysosomal engulfment of autophagosomes independent of ESCRT.
Small Molecule-Induced Alterations of Protein Polyubiquitination Revealed by Mass-Spectrometric Ubiquitome Analysis.
Large-scale purifications reveal yeast and human stress granule cores are heterogeneous particles with complex transcriptomes and proteomes.
Proteome-wide ligandability maps of drugs with diverse cysteine-reactive chemotypes.
PERK Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation.
USP37 protects mammalian cells during DNA replication stress by counteracting CUL2LRR1 and TRAIP.
A robust multiplex-DIA workflow profiles protein turnover regulations associated with cisplatin resistance and aneuploidy.
Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid.
The ER-associated Degradation Adaptor SEL1L is Dispensable for ER Homeostasis and the Differentiation of Spermatogenic Cells.
Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy.
Tunable metastability of condensates reconciles their dual roles in amyloid fibril formation.
RETREG1-mediated reticulophagy is activated by an ATF4-CEBPG/C/EBPγ heterodimer and confers protection against lipotoxicity.
Neuronal potassium channel activity triggers initiation of mRNA translation through binding of translation regulators.
KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions.
Machine learning to predict de novo protein-protein interactions.
Cytoplasmic DIS3 is an exosome-independent endoribonuclease with catalytic activity toward circular RNAs.
Sphingosine-1-phosphate signalling activates E-Syt1 to facilitate HDL-derived cholesterol transport.
Oxr1 and Ncoa7 regulate V-ATPase to achieve optimal pH for glycosylation within the Golgi apparatus and trans-Golgi network.
In vivo structure profiling reveals human cytosolic and mitochondrial tRNA structurome and interactome in response to stress.
Formation of giant ER sheets by pentadecanoic acid causes lipotoxicity in fission yeast.
Efficient cell-free translation from diverse human cell types.
Structural basis of ubiquitin ligase Nedd4-2 autoinhibition and regulation by calcium and 14-3-3 proteins.
Depletion-dependent activity-based protein profiling using SWATH/DIA-MS detects serine hydrolase lipid remodeling in lung adenocarcinoma progression.
Ribosomal expansion segment contributes to translation fidelity via N-terminal processing of ribosomal proteins.
The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance.
De novo assembly of nuclear stress bodies rearranges and enhances NFIL3 to restrain acute inflammatory responses.
hu.MAP3.0: atlas of human protein complexes by integration of >25,000 proteomic experiments.
Restoring calcium crosstalk between ER and mitochondria promotes intestinal stem cell rejuvenation through autophagy in aged Drosophila.
An animal toxin-antidote system kills cells by creating a novel cation channel.
Clustering of NLRP3 induced by membrane or protein scaffolds promotes inflammasome assembly.
TRIM37 prevents ectopic spindle pole assembly by peptide motif recognition and substrate-dependent oligomerization.
Predicting expression-altering promoter mutations with deep learning.
Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.
Structural insights into lipid membrane binding by human ferlins.
Tissue-specific SEC31A alternative splicing is regulated by RBM47 and controls lipid transport.
Mesoscale regulation of microtubule-organizing centers by the E3 ligase TRIM37.
RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α.
Structure-informed design of an ultrabright RNA-activated fluorophore.

Autophagy. 2025 May 25.

Endoplasmic reticulum tubule junctions are sites of autophagy.

Susan Ferro-Novick.

  Selective endoplasmic reticulum (ER) macroautophagy/autophagy, also called reticulophagy, is a disposal pathway that degrades ER domains. A major role of reticulophagy is the removal of ER domains that contain misfolded proteins resistant to ER-associated degradation (ERAD). Our studies have shown that RTN3L, the SEC24C-SEC23 COPII coat subcomplex, and the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L targets ERAD-resistant misfolded protein condensates for degradation at ER-reticulophagy sites (ERPHS), autophagic sites that form at tubule junctions. Unexpectedly, we found that the Parkinson disease protein PINK1 regulates ER tubulation. Loss of PINK1 disrupts the formation of peripheral tubule junctions, and, as a consequence, reticulophagy is blocked and misfolded proteins accumulate in the ER. Overexpression of the ER tubulating domain of DNM1L/DRP1, a multifunctional PINK1 kinase substrate that localizes to ER-mitochondria contact sites, increases junctions and restores reticulophagy. Our findings show that PINK1 shapes the ER to target misfolded proteins for RTN3L-SEC24C-mediated macroreticulophagy at defined ER sites, peripheral tubule junctions.

Keywords:  ER junctions; ER quality control; Reticulophagy

DOI:  https://doi.org/10.1080/15548627.2025.2508934
Nat Struct Mol Biol. 2025 May 26.

TRIP12 structures reveal HECT E3 formation of K29 linkages and branched ubiquitin chains.

Samuel A Maiwald, Laura A Schneider, Ronnald Vollrath, Joanna Liwocha, Matthew D Maletic, Kirby N Swatek, Monique P C Mulder, Brenda A Schulman.

Regulation by ubiquitin depends on E3 ligases forging chains of specific topologies, yet the mechanisms underlying the generation of atypical linkages remain largely elusive. Here we utilize biochemistry, chemistry, and cryo-EM to define the catalytic architecture producing K29 linkages and K29/K48 branches for the human HECT E3 TRIP12. TRIP12 resembles a pincer. One pincer side comprises tandem ubiquitin-binding domains, engaging the proximal ubiquitin to direct its K29 towards the ubiquitylation active site, and selectively capturing a distal ubiquitin from a K48-linked chain. The opposite pincer side-the HECT domain-precisely juxtaposes the ubiquitins to be joined, further ensuring K29 linkage specificity. Comparison to the prior structure visualizing K48-linked chain formation by UBR5 reveals a similar mechanism shared by two human HECT enzymes: parallel features of the E3s, donor and acceptor ubiquitins configure the active site around the targeted lysine, with E3-specific domains buttressing the acceptor for linkage-specific polyubiquitylation.

DOI: https://doi.org/10.1038/s41594-025-01561-1
Biochem Soc Trans. 2025 May 28. pii: BST20253034. [Epub ahead of print]

Insights into the role of collided ribosomes during the activation of the integrated stress response.

Ankanahalli N Nanjaraj Urs, Lucas Kim, Hani S Zaher.

  Mechanisms that regulate and reprogram gene expression are particularly important under stress conditions. The integrated stress response (ISR) signaling pathway is one such pro-survival and adaptive mechanism conserved in eukaryotes. The ISR is characterized by the activation of protein kinases that phosphorylate the eukaryotic initiation factor 2α (eIF2α) in response to several stress conditions, including nutrient deprivation, viral infection, and protein misfolding. Phosphorylation of eIF2α results in global inhibition of translation, while promoting the translation of a few pro-survival genes. Here, we focus on the mechanism of activation of the eIF2α kinase general control nonderepressible 2 (Gcn2). The protein was initially discovered in yeast more than four decades ago, and it was proposed to respond to amino acid starvation through the accumulation of deacylated tRNAs. However, more recent studies have changed our understanding of its activation and suggest a direct role for ribosome stalling and collisions in the process. In this review, we discuss the classical model for the tRNA-mediated activation of GCN2 and the recent shift in this model to accommodate the observations that wide-ranging translational stresses trigger its activation.

Keywords:  Gcn2; Gcn4; collided ribosomes; ribosome quality control; the integrated stress response; translational control

DOI:  https://doi.org/10.1042/BST20253034
Cell Rep. 2025 May 22. pii: S2211-1247(25)00507-8. [Epub ahead of print]44(6): 115736

The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.

Zaw Min Htet, Ken C Dong, Andreas Martin.

  The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. Besides targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by modulating proteasome conformational switching, yet the underlying mechanisms are unknown. Here, we use biochemical, mutational, and single-molecule fluorescence resonance energy transfer (FRET)-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin receptor subunit Rpn10, thereby stabilizing the proteasome's engagement-competent state and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to 4-fold faster turnover by the proteasome.

Keywords:  26S proteasome; ATP-dependent protein degradation; CP: Molecular biology; Rpn11 deubiquitinase; allosteric regulation; proteasome conformational dynamics; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2025.115736
RNA. 2025 May 29. pii: rna.080399.125. [Epub ahead of print]

Human CCR4 deadenylase homolog Angel1 is a Non-Stop mRNA Decay factor.

Tim Nicholson-Shaw, Megan E Dowdle, Yasmeen Ajaj, Mark Perelis, Amit Fulzele, Gene W Yeo, Eric J Bennett, Jens Lykke-Andersen.

  Translation elongation stalls trigger mRNA decay and degradation of the nascent polypeptide via translation-dependent quality control pathways. One such pathway, non-stop mRNA decay (NSD), targets aberrant mRNAs that lack stop codons for example due to premature polyadenylation. Here we identify Angel1, a CCR4 deadenylase homolog whose biochemical activity remains poorly defined, as a rate-limiting factor for NSD in human cells. Angel1 associates with mRNA coding regions and proteins involved in ribosome-associated quality control and mRNA decay, consistent with a factor that monitors translation elongation stalls. Depletion of Angel1 causes stabilization of reporter mRNAs that are targeted for NSD by the absence of stop codons, but not an mRNA targeted for nonsense-mediated decay. A conserved catalytic residue of Angel1 is critical for its function in NSD. Our findings identify Angel1 as a human NSD factor and suggest that Angel1 catalytic activity plays a critical role in the NSD pathway.

Keywords:  Angel1; No-Go Decay; Non-Stop Decay; Ribosome Quality Control; mRNA turnover

DOI:  https://doi.org/10.1261/rna.080399.125
Nature. 2025 May 28.

EndoMAP.v1 charts the structural landscape of human early endosome complexes.

Miguel A Gonzalez-Lozano, Ernst W Schmid, Enya Miguel Whelan, Yizhi Jiang, Joao A Paulo, Johannes C Walter, J Wade Harper.

Early or sorting endosomes are dynamic organelles that play key roles in proteome control by triaging plasma membrane proteins for either recycling or degradation in the lysosome1,2. These events are coordinated by numerous transiently associated regulatory complexes and integral membrane components that contribute to organelle identity during endosome maturation3. Although a subset of the several hundred protein components and cargoes known to associate with endosomes have been studied at the biochemical and/or structural level, interaction partners and higher-order molecular assemblies for many endosomal components remain unknown. Here, we combine crosslinking and native gel mass spectrometry4-7 of purified early endosomes with AlphaFold8,9 and computational analysis to create a systematic human endosomal structural interactome. We present 229 structural models for endosomal protein pairs and additional higher-order assemblies supported by experimental crosslinks from their native subcellular context, suggesting structural mechanisms for previously reported regulatory processes. Using induced neurons, we validate two candidate complexes whose interactions are supported by crosslinks and structural predictions: TMEM230 as a subunit of ATP8 and ATP11 lipid flippases10 and TMEM9 and TMEM9B as subunits of the chloride-proton antiporters CLCN3, CLCN4 and CLCN5 (ref. 11). This resource and its accompanying structural network viewer provide an experimental framework for understanding organellar structural interactomes and large-scale validation of structural predictions.

DOI: https://doi.org/10.1038/s41586-025-09059-y
Biochem Biophys Res Commun. 2025 May 21. pii: S0006-291X(25)00774-0. [Epub ahead of print]772 152060

Endolysosomal engulfment of autophagosomes independent of ESCRT.

Rui Tian, Zhe Wu, Yufen Wang, Chuangpeng Li, Fengping Liu, Yueguang Rong.

  Endolysosomes, considered the cellular recycling compartments, receive and degrade materials from multiple pathways. However, whether endolysosomes can acquire cargo through alternative mechanisms remains unclear. Here, we identify a previously unrecognized endolysosomal pathway for material uptake. In this process, endolysosomes extend two membrane protrusions that envelop and ultimately engulf autophagosomes, independently of autophagosome-endolysosome fusion and the endosomal sorting complex required for transport complex (ESCRT)-mediated microautophagy. The endolysosomes containing internalized autophagosomes, acquire additional autophagosomes through homotypic fusion. A subset of autophagosomes is marked by F-actin on their membranes and the majority of them contain the ER protein Sec61β and the peroxisomal protein Pex16 within their lumens, whereas mitochondria remain excluded. Our discovery of this endolysosomal process unveils a previously uncharacterized pathway for cargo acquisition by endolysosomes.

Keywords:  Autophagosomes; Endolysosomes

DOI:  https://doi.org/10.1016/j.bbrc.2025.152060
Angew Chem Int Ed Engl. 2025 May 30. e202508916

Small Molecule-Induced Alterations of Protein Polyubiquitination Revealed by Mass-Spectrometric Ubiquitome Analysis.

Siska Führer, Kai Gallant, Farnusch Kaschani, Markus Kaiser, Petra Janning, Herbert Waldmann, Malte Gersch.

  Small molecules which alter protein ubiquitination are emerging as therapeutics due to their ability to modulate targets previously deemed undruggable. These compounds comprise PROTACs, molecular glue degraders and DUB inhibitors, among others. However, methods for the proteome-wide monitoring of compound-induced changes in protein polyubiquitination, which may also detect non-degradative modifications, are lacking. Here, we report the utilization of polyubiquitin enrichment coupled to mass spectrometry to monitor small molecule-induced changes in cellular protein ubiquitination. We established enrichment through tandem ubiquitin binding entities (TUBEs) following semi-denaturing cell lysis and devised an elution protocol compatible with downstream LC-MS/MS analysis. We demonstrate broad applicability of the workflow by assessing ubiquitination changes induced by a PROTAC, a p97 inhibitor and deubiquitinase inhibitors. Application of the assay to compounds inhibiting the deubiquitinase USP7 revealed the induction of non-degradative ubiquitination on the UBE3A E3 ligase. Collectively, we established a versatile proteomics method to facilitate the direct investigation of cellular polyubiquitination, with high relevance for the identification and characterization of protein degraders, stabilizers and other molecules with ubiquitin-mediated bioactivity.

Keywords:  Biological activity; Drug Discovery; Protein modifications; Ubiquitin proteasome system; proteomics

DOI:  https://doi.org/10.1002/anie.202508916
Cell Rep. 2025 May 23. pii: S2211-1247(25)00509-1. [Epub ahead of print]44(6): 115738

Large-scale purifications reveal yeast and human stress granule cores are heterogeneous particles with complex transcriptomes and proteomes.

Natalia A Demeshkina, Adrian R Ferré-D'Amaré.

  Stress granules are a conserved response of eukaryotic cells to environmental insults. These cytoplasmic ribonucleoprotein condensates have hitherto been primarily studied by microscopy, which showed previously that they comprise dense ∼200 nm cores embedded in a diffuse shell. We have developed large-scale purifications of budding yeast and mammalian (HEK293T cell) stress granule cores that do not rely on immunoprecipitation of candidate protein constituents. These unbiased preparations reveal that stress granule cores are discrete particles with variable size (average, 135 and 225 nm for yeast and human, respectively) and shape. Proteomics and transcriptomics demonstrate complex composition. The results of hybridization chain reaction fluorescence in situ hybridization (FISH) analyses in HEK293T cells are consistent with stress granule cores having heterogeneous composition, i.e., each stress granule core particle contains only a limited number of mRNA species. Biochemical purification now opens the way to mechanistic analysis of the heterogeneity and complexity of stress granules.

Keywords:  CP: Molecular biology; cytoplasmic condensate; endoplasmic reticulum; mitochondria; oxidative stress; proteomics; ribonucleoprotein; size-exclusion chromatography; sucrose density gradient centrifugation; transcriptomics

DOI:  https://doi.org/10.1016/j.celrep.2025.115738
Nat Commun. 2025 May 26. 16(1): 4863

Proteome-wide ligandability maps of drugs with diverse cysteine-reactive chemotypes.

Caiping Tian, Lu Sun, Keke Liu, Ling Fu, Yi Zhang, Wendong Chen, Fuchu He, Jing Yang.

Covalent drug discovery has experienced a revival since the 2013 approval of the first cysteine-targeting kinase inhibitors. Many drugs that were discovered by serendipity also possess the ability to react with cysteine residues, leading to interactions with multiple proteins. This widespread interaction, known as promiscuity, necessitates a comprehensive study of how these drugs engage with cysteines throughout the proteome. Here we report a large-scale analysis to meet this need by defining proteome-wide cysteine ligandability maps of 70 drugs in native biological systems. We examined over 24,000 cysteines in the human proteome, pinpointing 279 proteins as potential drug targets across diverse functional categories. We further validated several cysteine engagement events, uncovering previously unknown cysteine sites on both established drug targets and proteins that are generally difficult to address with small molecules. Additionally, our findings highlighted an opportunity to harness a drug-cysteine interaction for targeted protein degradation. Together, our analysis provides an invaluable resource for advancing the development of covalent drugs.

DOI: https://doi.org/10.1038/s41467-025-60068-x
Blood. 2025 May 28. pii: blood.2024027846. [Epub ahead of print]

PERK Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation.

Manxi Zheng, Qinlu Peng, Erin M Kropp, Zhejuan Shen, Suxuan Liu, Zhengyou Yin, Sho Matono, Takao Iwawaki, Xiang Wang, Ken Inoki, Yang Mei, Qing Li, Lu Liu.

The integrity of the hematopoietic stem cell (HSC) pool depends on effective long-term self-renewal and the timely elimination of damaged or differentiation-prone HSCs. While the PERK branch of the unfolded protein response (UPR) has been shown to initiate pro-apoptotic signaling in response to ER stress in vitro, its role in regulating HSC fate in vivo remains incompletely understood. Here, we demonstrate that PERK is dispensable for steady-state hematopoiesis and HSC self-renewal under homeostatic conditions. However, under ER stress induced by disruption of ER-associated degradation (ERAD), via knockout of key components such as Sel1L or Hrd1, PERK becomes activated and drives HSC proliferation and depletion. Notably, deletion of PERK or expression of a kinase-dead PERK mutant significantly rescues the HSC defects caused by Sel1L or Hrd1 loss. Mechanistically, ERAD deficiency does not lead to increased HSC apoptosis or elevated reactive oxygen species (ROS), and PERK knockout has minimal impact on HSC apoptosis. Instead, PERK activation promotes aberrant mTOR signaling and HSC hyperproliferation, ultimately compromising self-renewal capacity. This PERK-driven elimination of stressed HSCs may function as a protective mechanism to maintain overall HSC pool integrity. Collectively, our findings reveal a previously unrecognized, proliferative, and apoptosis-independent role for PERK in regulating HSC fate under ER stress, highlighting a novel mechanism for preserving HSC homeostasis.

DOI: https://doi.org/10.1182/blood.2024027846
Cell Rep. 2025 May 22. pii: S2211-1247(25)00510-8. [Epub ahead of print]44(6): 115739

USP37 protects mammalian cells during DNA replication stress by counteracting CUL2LRR1 and TRAIP.

Fabrizio Villa, Johanna Ainsworth, Karim P M Labib.

  The USP37 deubiquitylase is essential for mammalian cells to survive DNA replication stress, but the underlying mechanisms are unknown. Here, we demonstrate that USP37 binds the CDC45-MCM-GINS (CMG) helicase, which forms the stable core of the replisome until DNA replication termination when CMG is ubiquitylated and disassembled. USP37 contacts CDC45, and structure-guided mutations that displace USP37 from CMG cause sensitivity to DNA synthesis defects or topological stress. Binding to CDC45 at replication forks enables USP37 to counteract CMG ubiquitylation by the CUL2LRR1 ligase, which subsequently induces replisome disassembly during termination. Correspondingly, depletion of CUL2LRR1 suppresses the sensitivity of Usp37 mutants to DNA synthesis defects and ATR checkpoint kinase inhibitors. In contrast, mutation of the TRAIP ubiquitin ligase specifically suppresses the sensitivity of Usp37 mutants to topological stress. We propose that USP37 protects mammalian cells from replication stress by reversing the untimely action of the CUL2LRR1 and TRAIP ubiquitin ligases.

Keywords:  ATR checkpoint kinase; CMG helicase; CP: Molecular biology; CUL2(LRR1); DNA replication; TRAIP; USP37; deubiquitylase; ubiquitylation

DOI:  https://doi.org/10.1016/j.celrep.2025.115739
Nat Commun. 2025 May 30. 16(1): 5034

A robust multiplex-DIA workflow profiles protein turnover regulations associated with cisplatin resistance and aneuploidy.

Barbora Salovska, Wenxue Li, Oliver M Bernhardt, Pierre-Luc Germain, Qinyue Wang, Tejas Gandhi, Lukas Reiter, Yansheng Liu.

Quantifying protein turnover is fundamental to understanding cellular processes and advancing drug discovery. Multiplex-DIA mass spectrometry (MS), combined with dynamic SILAC labeling (pulse-SILAC, or pSILAC) reliably measures protein turnover and degradation kinetics. Previous multiplex-DIA-MS workflows have employed various strategies including leveraging the highest isotopic labeling channels to enhance the detection of isotopic signal pairs. Here we present a robust workflow that integrates a machine learning algorithm and channel-specific statistical filtering, enabling dynamic adaptation to channel ratio changes across multiplexed experiments and enhancing both coverage and accuracy of protein turnover profiling. We also introduce KdeggeR, a data analysis tool optimized for pSILAC-DIA experiments, which determines and visualizes peptide and protein degradation profiles. Our workflow is broadly applicable, as demonstrated on 2-channel and 3-channel DIA datasets and across two MS platforms. Applying this framework to an aneuploid cancer cell model before and after cisplatin resistance, we uncover strong proteome buffering of key protein complex subunits encoded by the aneuploid genome mediated by protein degradation. We identify resistance-associated turnover signatures, including mitochondrial metabolic adaptation via accelerated degradation of respiratory complexes I and IV. Our approach provides a powerful platform for high-throughput, quantitative analysis of proteome dynamics and stability in health and disease.

DOI: https://doi.org/10.1038/s41467-025-60319-x
Elife. 2025 May 30. pii: RP93621. [Epub ahead of print]13

Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid.

Josep Fita-Torró, José Luis Garrido-Huarte, Lucía López-Gil, Agnès H Michel, Benoit Kornmann, Amparo Pascual-Ahuir, Markus Proft.

  Mitochondria-mediated cell death is critically regulated by bioactive lipids derived from sphingolipid metabolism. The lipid aldehyde trans-2-hexadecenal (t-2-hex) induces mitochondrial dysfunction from yeast to humans. Here, we apply unbiased transcriptomic, functional genomics, and chemoproteomic approaches in the yeast model to uncover the principal mechanisms and biological targets underlying this lipid-induced mitochondrial inhibition. We find that loss of Hfd1 fatty aldehyde dehydrogenase function efficiently sensitizes cells for t-2-hex inhibition and apoptotic cell death. Excess of t-2-hex causes a profound transcriptomic response with characteristic hallmarks of impaired mitochondrial protein import, like activation of mitochondrial and cytosolic chaperones or proteasomal function and severe repression of translation. We confirm that t-2-hex stress induces rapid accumulation of mitochondrial pre-proteins and protein aggregates and subsequent activation of Hsf1- and Rpn4-dependent gene expression. By saturated transposon mutagenesis, we find that t-2-hex tolerance requires an efficient heat shock response and specific mitochondrial and ER functions and that mutations in ribosome, protein, and amino acid biogenesis are beneficial upon t-2-hex stress. We further show that genetic and pharmacological inhibition of protein translation causes t-2-hex resistance, indicating that loss of proteostasis is the predominant consequence of the pro-apoptotic lipid. Several TOM subunits, including the central Tom40 channel, are lipidated by t-2-hex in vitro and mutation of accessory subunits Tom20 or Tom70 confers t-2-hex tolerance. Moreover, the Hfd1 gene dose determines the strength of t-2-hex mediated inhibition of mitochondrial protein import, and Hfd1 co-purifies with Tom70. Our results indicate that the transport of mitochondrial precursor proteins through the outer mitochondrial membrane is sensitively inhibited by the pro-apoptotic lipid and thus represents a hotspot for pro- and anti-apoptotic signaling.

Keywords:  S. cerevisiae; apoptosis; biochemistry; chemical biology; genetics; genomics; lipid signaling; mitochondrial protein import; proteostasis; sphingolipid metabolism; yeast

DOI:  https://doi.org/10.7554/eLife.93621
J Biol Chem. 2025 May 22. pii: S0021-9258(25)02133-7. [Epub ahead of print] 110283

The ER-associated Degradation Adaptor SEL1L is Dispensable for ER Homeostasis and the Differentiation of Spermatogenic Cells.

Nusrat Jahan Tushi, Zhibing Zhang, Shengyi Sun.

  The SEL1L-HRD1 complex is a critical component of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, essential for maintaining ER homeostasis and cellular function. While the crucial roles of SEL1L and HRD1 in various physiological processes have been reported in mice and humans, their specific functions in male germ cells remain unexplored. Here, we show that, while SEL1L is highly expressed in spermatogenic cells, it is dispensable for their differentiation and ER homeostasis. SEL1L deletion in these cells does not affect sperm count, motility, male fertility, or testicular histology. Mechanistically, our data show that SEL1L loss reduces HRD1 protein levels in spermatids but unexpectedly, not in spermatocytes. Furthermore, SEL1L deficiency does not induce overt ER stress response, ER dilation, or cell death in the testes. Collectively, these findings indicate that SEL1L is not required for ER homeostasis or the differentiation of male germ cells.

Keywords:  ER stress; ERAD; SEL1L; endoplasmic reticulum; sperm; spermatogenesis; spermiogenesis

DOI:  https://doi.org/10.1016/j.jbc.2025.110283
Autophagy. 2025 May 25.

Evidence of an unprecedented cytoplasmic function of DDX11, the Warsaw breakage syndrome DNA helicase, in regulating autophagy.

Raffaella Bonavita, Antonello Prodomo, Giuseppe Cortone, Fulvia Vitale, Marcello Germoglio, Angeleen Fleming, Jesper A Balk, Job De Lange, Maurizio Renna, Francesca M Pisani.

  DDX11 is a DNA helicase involved in critical cellular functions, including DNA replication/repair/recombination as well as sister chromatid cohesion establishment. Bi-allelic mutations of DDX11 lead to Warsaw breakage syndrome (WABS), a rare genome instability disorder marked by significant prenatal and postnatal growth restriction, microcephaly, intellectual disability, and sensorineural hearing loss. The molecular mechanisms underlying WABS remain largely unclear. In this study, we uncover a novel role of DDX11 in regulating the macroautophagic/autophagic pathway. Specifically, we demonstrate that knockout of DDX11 in RPE-1 cells hinders the progression of autophagy. DDX11 depletion significantly reduces the conversion of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), suggesting a defect in autophagosome biogenesis. This is supported by imaging analysis with a LC3 reporter fused in tandem with the red and green fluorescent proteins (mRFP-GFP-LC3), which reveals fewer autophagosomes and autolysosomes in DDX11-knockout cells. Moreover, the defect in autophagosome biogenesis, observed in DDX11-depleted cells, is linked to an upstream impairment of the ATG16L1-precursor trafficking and maturation, a step critical to achieve the LC3 lipidation. Consistent with this, DDX11-lacking cells exhibit a diminished capacity to clear aggregates of a mutant HTT (huntingtin) N-terminal fragment fused to the green fluorescent protein (HTTQ74-GFP), an autophagy substrate. Finally, we demonstrate the occurrence of a functional interplay between DDX11 and SQSTM1, an autophagy cargo receptor protein, in supporting LC3 modification during autophagosome biogenesis. Our findings highlight a novel unprecedented function of DDX11 in the autophagy process with important implications for our understanding of WABS etiology.

Keywords:  Autophagosome biogenesis; DDX11; DNA helicase; LC3; SQSTM1/p62; Warsaw breakage syndrome; neurodevelopmental disorders

DOI:  https://doi.org/10.1080/15548627.2025.2507617
Mol Cell. 2025 May 23. pii: S1097-2765(25)00416-2. [Epub ahead of print]

Tunable metastability of condensates reconciles their dual roles in amyloid fibril formation.

Tapojyoti Das, Fatima K Zaidi, Mina Farag, Kiersten M Ruff, Tharun Selvam Mahendran, Anurag Singh, Xinrui Gui, James Messing, J Paul Taylor, Priya R Banerjee, Rohit V Pappu, Tanja Mittag.

  Stress granules form via co-condensation of RNA-binding proteins (RBPs) containing prion-like low-complexity domains (PLCDs) with RNA molecules. Homotypic interactions among PLCDs can drive amyloid fibril formation that is enhanced by amyotrophic lateral sclerosis (ALS)-associated mutations. We report that condensation- versus fibril-driving homotypic interactions are separable for A1-LCD, the PLCD of hnRNPA1. These separable interactions lead to thermodynamically metastable condensates and globally stable fibrils. Interiors of condensates suppress fibril formation, whereas interfaces have the opposite effect. ALS-associated mutations enhance the stability of fibrils and weaken condensate metastability, thus enhancing the rate of fibril formation. We designed mutations to enhance A1-LCD condensate metastability and discovered that stress granule disassembly in cells can be restored even when the designed variants carry ALS-causing mutations. Therefore, fibril formation can be suppressed by condensate interiors that function as sinks. Condensate sink potentials are influenced by their metastability, which is tunable through separable interactions even among minority components of stress granules.

Keywords:  RNP granule; amyotrophic lateral sclerosis; fibril formation; frontotemporal dementia; metastability; phase separation; prion-like domain; sink potential; stress granule; supersaturation

DOI:  https://doi.org/10.1016/j.molcel.2025.05.011
Autophagy. 2025 May 28.

RETREG1-mediated reticulophagy is activated by an ATF4-CEBPG/C/EBPγ heterodimer and confers protection against lipotoxicity.

Suwei Jin, Mingzhu Yan, Yongguang Liu, Shanshan Zhang, Hongbin Song, Chenxi Cao, Yujia Li, Guibo Sun, Linhu Ye, Jianzhi Chen, Wen Han, Lingyu Li, Qi Chang.

  Excessive fatty acid triggers endoplasmic reticulum (ER) stress, leading to lipotoxicity, which plays a vital role in the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). Reticulophagy is recently identified as an integral process in maintaining ER homeostasis during ER stress. However, our knowledge of reticulophagy in lipotoxicity remains limited, and the underlying molecular mechanisms are unclear. Here we showed that mild, short-term lipotoxicity induced by palmitic acid stimulated reticulophagy in vitro, mediated primarily by the selective receptor RETREG1. Knockdown of RETREG1 in HepG2 cells and primary hepatocytes exacerbated palmitic acid-induced cell damage and death. Having demonstrated the indispensability of ATF4 and CEBPG/C/EBPγ in transcriptional upregulation of RETREG1, we found that ATF4 forms a heterodimer with CEBPG and identified their binding sites in the promoter and enhancer regions of RETREG1 gene. In mice with acute hepatic lipotoxicity, RETREG1-mediated reticulophagy was activated, conferring protection against liver injury, as retreg1 knockout mice exhibited more severe liver injury than wild-type mice. In contrast, reticulophagy initiation was defective in a high fat diet-induced mouse model of MASLD, possibly due to decreased gene expression of Retreg1 driven by the suppression in ATF4 and CEBPG. Our study underscores the crucial role of RETREG1-mediated reticulophagy, which is co-regulated by ATF4 and CEBPG, in response to lipotoxicity, suggesting that activation of reticulophagy may represent a strategy against MASLD.

Keywords:  ATF4; CEBPG; MASLD; RETREG1; lipotoxicity; reticulophagy

DOI:  https://doi.org/10.1080/15548627.2025.2512884
Sci Adv. 2025 May 30. 11(22): eadv3140

Neuronal potassium channel activity triggers initiation of mRNA translation through binding of translation regulators.

Taylor J Malone, Jing Wu, Yalan Zhang, Pawel Licznerski, Rongmin Chen, Sheikh Nahiyan, Maysam Pedram, Elizabeth A Jonas, Leonard K Kaczmarek.

Neuronal activity stimulates mRNA translation crucial for learning and development, but the mechanism linking translation to neuronal activity is not understood. In humans, learning and memory are severely disrupted by mutations in the potassium channel Slack (KCNT1, Slo2.2). We find that pharmacological stimulation of this channel and a constitutively active Slack mutation stimulate mRNA translation of a reporter for β-actin mRNA in cell lines and increases the synthesis of β-actin in the neurites of cortical neurons. Moreover, channel activation promotes the binding of two key mRNA translation regulators, FMRP (fragile X mental retardation protein) and CYFIP1 (cytoplasmic FMR1-interacting protein 1), to the channel itself, releasing both from eIF4E (eukaryotic initiation factor 4E), where they normally inhibit initiation of translation. This interaction provides a molecular mechanism for Slack activity-dependent regulation of translation and suggests that the effects of Slack mutations on this process may explain the severe intellectual disabilities associated with these mutations.

DOI: https://doi.org/10.1126/sciadv.adv3140
EMBO Rep. 2025 May 28.

KEAP1 retention in phase-separated p62 bodies drives liver damage under autophagy-deficient conditions.

Shuhei Takada, Nozomi Shinomiya, Gaoxin Mao, Hikaru Tsuchiya, Tomoaki Koga, Satoko Komatsu-Hirota, Yu-Shin Sou, Manabu Abe, Elena Ryzhii, Michitaka Suzuki, Mitsuyoshi Nakao, Satoshi Waguri, Hideaki Morishita, Masaaki Komatsu.

  Phase-separated p62 bodies activate NRF2, a key transcription factor for antioxidant response, by sequestering KEAP1, which targets NRF2 for degradation. Although p62 bodies containing KEAP1 are degraded by autophagy, they accumulate in various liver disorders. Their precise disease role remains unclear. We show that excessive KEAP1 retention in p62 bodies and NRF2 activation are major causes of liver damage when autophagy is impaired. In mice with weakened or blocked p62-KEAP1 interactions, KEAP1 retention and NRF2 activation under autophagy-deficient conditions were suppressed. Transcriptome and proteome analyses reveal that p62 mutants unable to bind KEAP1 normalize the expression of NRF2 targets induced by defective autophagy. Autophagy deficiency causes organelle accumulation, especially of the ER, regardless of p62 mutation. Liver damage and hepatomegaly resulting from autophagy suppression markedly improved in mice carrying p62 mutants, particularly those with blocked KEAP1 binding. These findings highlight excessive KEAP1 retention in p62 bodies and defective organelle turnover as key drivers of liver pathology, underscoring the significance of phase separation in vivo.

Keywords:  KEAP1; Liquid–Liquid Phase Separation; NRF2; Stress Response; p62

DOI:  https://doi.org/10.1038/s44319-025-00483-9
Trends Biotechnol. 2025 May 26. pii: S0167-7799(25)00158-1. [Epub ahead of print]

Machine learning to predict de novo protein-protein interactions.

Pablo Gainza, Richard D Bunker, Sharon A Townson, John C Castle.

  Advances in machine learning for structural biology have dramatically enhanced our capacity to predict protein-protein interactions (PPIs). Here, we review recent developments in the computational prediction of PPIs, particularly focusing on innovations that enable interaction predictions that have no precedence in nature, termed de novo. We discuss novel machine learning algorithms for PPI prediction, including approaches based on co-folding and atomic graphs. We further highlight methods that learn from molecular surfaces, which can predict PPIs not found in nature including interactions induced by small molecules. Finally, we explore the emerging biotechnological applications enabled by these predictive capabilities, including the prediction of antibody-antigen complexes and molecular glue-induced PPIs, and discuss their potential to empower drug discovery and protein engineering.

Keywords:  machine learning; molecular glues; molecular surfaces; protein interaction design; protein–protein interactions (PPIs)

DOI:  https://doi.org/10.1016/j.tibtech.2025.04.013
Cell Rep. 2025 May 28. pii: S2211-1247(25)00540-6. [Epub ahead of print]44(6): 115769

Cytoplasmic DIS3 is an exosome-independent endoribonuclease with catalytic activity toward circular RNAs.

Claudia Latini, Julian Eichlinger, Anna-Lisa Fuchs, Si-Nan Zhai, Hung Ho-Xuan, Gerhard Lehmann, Petar Glažar, Nikolaus Rajewsky, Astrid Bruckmann, Li Yang, Remco Sprangers, Gunter Meister.

  The ribonuclease DIS3 interacts through its PIN domain with the nuclear exosome and degrades linear RNA substrates using its exoribonuclease domain. However, the PIN domain is also an active endoribonuclease, but cellular substrates are largely unknown. Here, we use a biochemical strategy to find ribonucleases that could degrade circular RNAs (circRNAs). Due to the lack of accessible ends, circRNAs are resistant to exonucleolytic cleavage and are thus more stable than linear RNAs. Using biochemical assays, we identify DIS3 as a candidate for circRNA degradation and demonstrate that it partially resides in the cytoplasm, where circRNAs are degraded. DIS3 shows cleavage activity toward a number of circRNAs and functions independently of the exosome core in vitro. Upon knockdown of DIS3 in cell lines, selected circRNAs are moderately stabilized. We thus propose that cytoplasmic DIS3 functions as a stand-alone enzyme independently of the exosome core and may contribute to circRNA turnover.

Keywords:  CP: Molecular biology; DIS3; PIN domain; RNA metabolism; circular RNA; cytoplasm; decay; degradation; exosome

DOI:  https://doi.org/10.1016/j.celrep.2025.115769
Nat Cell Biol. 2025 May 28.

Sphingosine-1-phosphate signalling activates E-Syt1 to facilitate HDL-derived cholesterol transport.

Zizhen Xu, Ying Meng, Jonathan St-Germain, Arezoo Afshari, Charneal L Dixon, Saskia Heybrock, Qiang Zhao, Xialian Weng, Jishun Chen, Richard Collins, Hu Hu, Quan Zhou, Qiming Sun, Pinglong Xu, Wei Liu, Paul Saftig, Brian Raught, Gregory D Fairn, Dante Neculai.

Cholesterol derived from high-density lipoprotein (HDL) is rapidly redistributed to intracellular compartments in steroidogenic and bile-producing cells, but the molecular mechanisms governing this essential transport process remain poorly understood. Here we uncover a signalling cascade coordinating HDL-derived cholesterol transport through membrane contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM). We find that HDL-resident sphingosine-1-phosphate (S1P) activates S1P receptor 3 and its associated G protein αq, leading to phospholipase-C-β3-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate and an elevation in cytosolic calcium. This calcium signal triggers the rapid recruitment of Extended-Synaptotagmin 1 to ER-PM membrane contact sites. Genetic or pharmacological disruption of this pathway impairs the non-vesicular transfer of HDL-derived cholesterol to intracellular compartments. Our findings reveal how HDL binding to the cell surface alters ER-PM membrane contact site dynamics through S1P signalling. This ensures efficient offloading and redistribution of HDL cholesterol to support steroid and bile acid synthesis.

DOI: https://doi.org/10.1038/s41556-025-01665-2
Proc Natl Acad Sci U S A. 2025 Jun 03. 122(22): e2505975122

Oxr1 and Ncoa7 regulate V-ATPase to achieve optimal pH for glycosylation within the Golgi apparatus and trans-Golgi network.

Shin-Ichiro Yoshimura, Tomoaki Sobajima, Masataka Kunii, Akihiro Harada.

  Maintenance of pH within membranous organelles is crucial for cellular processes such as posttranslational modifications, ligand-receptor interactions, and proteostasis. The precise mechanisms that determine the luminal pH of each organelle are not fully understood. This study investigated the mechanisms that regulate luminal pH to ensure optimal enzymatic activity. We identified Oxr1 and its paralog Ncoa7, which regulate the vacuolar-type proton pump ATPase (V-ATPase) at the Golgi apparatus and trans-Golgi network (TGN). Oxr1 and Ncoa7 were predominantly localized at the Golgi and TGN membranes, dependent on their binding to various GTP-bound Rab proteins. In vitro experiments using purified recombinant proteins indicated that Oxr1 and Ncoa7 directly bind to the catalytic subunit of V-ATPase, inhibiting its ATP hydrolytic activity via their TLDc domains. We observed significant acidification of the Golgi/TGN lumen in Oxr1- and Ncoa7-depleted cells. Lectin blot analysis demonstrated that depletion of Oxr1 and Ncoa7 led to a defect in protein glycosylation, a major enzymatic posttranslational modification in the Golgi and TGN. Furthermore, depletion of Oxr1 and Ncoa7, along with drug-induced inhibition of glycosylation, increased lysosomal pH and sensitivity to silicon dioxide-induced membrane damage. This apparent lysosomal dysfunction suggested that, in addition to the Golgi and TGN, Oxr1 and Ncoa7 also contribute to the integrity of other organelles. Our findings indicate that Oxr1 and Ncoa7 protect the Golgi and TGN lumen from excess acidification by inhibiting V-ATPase activity and providing an optimal environment for enzymatic activity in the Golgi and TGN.

Keywords:  Golgi apparatus/Trans-Golgi network; Rab; V-ATPase; congenital disorders of glycosylation; glycosylation

DOI:  https://doi.org/10.1073/pnas.2505975122
Nat Commun. 2025 May 30. 16(1): 5041

In vivo structure profiling reveals human cytosolic and mitochondrial tRNA structurome and interactome in response to stress.

Noah Peña, Yichen Hou, Christopher P Watkins, Sihao Huang, Wen Zhang, Christopher D Katanski, Tao Pan.

Transfer RNA (tRNA) is the most abundant cellular RNA family in terms of copy numbers. It not only folds into defined structures but also has complex cellular interaction networks involving aminoacyl-tRNA synthetases, translation factors, and ribosomes. The human tRNAome is comprised of chromosomal-encoded tRNAs with a large sequence diversity and mitochondrial-encoded tRNAs with A/U-rich sequences and noncanonical tertiary interactions. How tRNA folding and interactions in a eukaryotic cell respond to stress is poorly understood. Here, we develop DM-DMS-MaPseq, which utilizes in vivo dimethyl-sulfate (DMS) chemical probing and mutational profiling (MaP) coupled with demethylase (DM) treatment in transcriptome-wide tRNA sequencing to profile structures and the cellular interactions of human chromosomal and mitochondrial-encoded tRNAs. We found that tRNAs maintain stable structures in vivo, but the in vivo DMS profiles are vastly different from those in vitro, which can be explained by their interactions with cellular proteins and the ribosome. We also identify cytosolic and mitochondrial tRNA structure and interaction changes upon arsenite treatment, a type of oxidative stress that induces translational reprogramming, which is consistent with global translation repression in both compartments. Our results reveal variations of tRNA structurome and dynamic interactome that have functional consequences in translational regulation.

DOI: https://doi.org/10.1038/s41467-025-59435-5
Proc Natl Acad Sci U S A. 2025 Jun 03. 122(22): e2422126122

Formation of giant ER sheets by pentadecanoic acid causes lipotoxicity in fission yeast.

Yojiro Hoshikawa, Natsuho Shirota, Hiroshi Tsugawa, Satoshi Kimura, Akihisa Matsuyama, Yoko Yashiroda, Hideaki Kakeya, Makoto Arita, Reiko Iizumi, Minoru Yoshida, Shinichi Nishimura.

  Excess amounts of saturated fatty acids (FAs) are toxic to organisms, a condition termed lipotoxicity, which is often accompanied by pleiotropic cellular and tissue dysfunction. Here, we show that pentadecanoic acid (C15:0) exerts toxicity on the fission yeast Schizosaccharomyces pombe by generating an aberrantly planar endoplasmic reticulum (ER) structure, which we named a "giant ER sheet." Untargeted lipidomics revealed that C15:0 is incorporated into complex lipids depending on an acyl-CoA ligase Lcf1 and an acyl-CoA transferase Slc1, thereby increasing the saturation level of the acyl chains. The toxicity and giant ER sheet formation were abolished by deleting lcf1 or slc1 gene, indicating that the incorporation of C15:0 into glycerophospholipids causes giant ER sheet formation. The giant ER sheets disrupted the correct migration of Mid1, a protein determining the cell division site, and physically blocked septum formation, hindering correct cell separation. Our results suggest that the ER is the primary site targeted by saturated FAs, leading to lipotoxicity.

Keywords:  endoplasmic reticulum; lipotoxicity; yeast

DOI:  https://doi.org/10.1073/pnas.2422126122
J Biol Chem. 2025 May 28. pii: S0021-9258(25)02157-X. [Epub ahead of print] 110307

Efficient cell-free translation from diverse human cell types.

Jana Ziegelmüller, Nikolaos Kouvelas, Nino Schwaller, Priyanka Thambythurai, Alexander M Hofer, Oliver Mühlemann, Evangelos D Karousis.

  Cell-free translation systems are indispensable for studying protein synthesis, enabling researchers to explore translational regulation across different cell types. The difficulties in producing cell-free translation systems from different cell types limit the ability to study regulatory mechanisms that depend on different biological contexts. Here, we present a scalable method for preparing translation-competent lysates from a range of frequently used human cell lines using dual centrifugation. We optimized lysis conditions for adherent and suspension cells, producing high-quality lysates from HEK-293 (adherent and in suspension), HeLa, SH-SY5Y, and U2OS cells. Our results demonstrate that cell-specific factors influence translation efficiency, with adherent HeLa cells showing the highest activity. We also observed that sensitivity to lysis conditions varies between cell lines, underscoring the importance of fine-tuning parameters for efficient protein production. Our method provides a robust and adaptable approach for generating cell-type-specific lysates, broadening the application of in vitro translation systems in studying translational mechanisms.

Keywords:  Cell-type-specific lysates; Dual centrifugation; In vitro translation; Protein synthesis; Translational regulation; cell-free translation; synthetic biology

DOI:  https://doi.org/10.1016/j.jbc.2025.110307
Nat Commun. 2025 May 26. 16(1): 4875

Structural basis of ubiquitin ligase Nedd4-2 autoinhibition and regulation by calcium and 14-3-3 proteins.

Masa Janosev, Dalibor Kosek, Andrej Tekel, Rohit Joshi, Karolina Honzejkova, Pavel Pohl, Tomas Obsil, Veronika Obsilova.

Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.

DOI: https://doi.org/10.1038/s41467-025-60207-4
Nat Commun. 2025 May 27. 16(1): 4889

Depletion-dependent activity-based protein profiling using SWATH/DIA-MS detects serine hydrolase lipid remodeling in lung adenocarcinoma progression.

Tatjana Sajic, Matej Vizovišek, Stephan Arni, Rodolfo Ciuffa, Martin Mehnert, Sébastien Lenglet, Walter Weder, Hector Gallart-Ayala, Julijana Ivanisevic, Marija Buljan, Aurelien Thomas, Sven Hillinger, Ruedi Aebersold.

Systematic inference of enzyme activity in human tumors is key to understanding cancer progression and resistance to therapy. However, standard protein or transcript abundances are blind to the activity status of the measured enzymes, regulated, for example, by active-site amino acid mutations or post-translational protein modifications. Current methods for activity-based proteome profiling (ABPP), which combine mass spectrometry (MS) with chemical probes, quantify the fraction of enzymes that are catalytically active. Here, we describe depletion-dependent ABPP (dd-ABPP) combined with automated SWATH/DIA-MS, which simultaneously determines three molecular layers of studied enzymes: i) catalytically active enzyme fractions, ii) enzyme and background protein abundances, and iii) context-dependent enzyme-protein interactions. We demonstrate the utility of the method in advanced lung adenocarcinoma (LUAD) by monitoring nearly 4000 protein groups and 200 serine hydrolases (SHs) in tumor and adjacent tissue sections routinely collected for patient histopathology. The activity profiles of 23 SHs and the abundance of 59 proteins associated with these enzymes retrospectively classified aggressive LUAD. The molecular signature revealed accelerated lipoprotein depalmitoylation via palmitoyl(protein)hydrolase activities, further confirmed by excess palmitate and its metabolites. The approach is universal and applicable to other enzyme families with available chemical probes, providing clinicians with a biochemical rationale for tumor sample classification.

DOI: https://doi.org/10.1038/s41467-025-59564-x
Nucleic Acids Res. 2025 May 22. pii: gkaf448. [Epub ahead of print]53(10):

Ribosomal expansion segment contributes to translation fidelity via N-terminal processing of ribosomal proteins.

Riku Nagai, Olivia L Milam, Tatsuya Niwa, William J Howell, Jacob A Best, Hideji Yoshida, Carver D Freeburg, John M Koomen, Kotaro Fujii.

Eukaryotic ribosomes exhibit higher mRNA translation fidelity than prokaryotic ribosomes, partly due to eukaryote-specific ribosomal RNA (rRNA) insertions. Among these, expansion segment 27L (ES27L) on the 60S subunit enhances fidelity by anchoring methionine aminopeptidase (MetAP) at the nascent protein exit tunnel, accelerating co-translational N-terminal initiator methionine (iMet) processing. However, the mechanisms by which iMet processing influences translation fidelity remain unknown. Using yeast in vitro translation (IVT) systems, we found that inhibiting co-translational iMet processing does not impact ribosome decoding of ongoing peptide synthesis. Instead, our novel method to monitor iMet processing in vivo revealed that ribosomes purified from strains lacking MetAP ribosomal association (ES27L Δb1-4) or major yeast MetAP (Δmap1) increase iMet retention on ribosomal proteins (RPs). Given the densely packed structure of ribosomes, iMet retention on RPs may distort ribosomal structure and impair its function. Indeed, reconstituted IVT systems containing iMet-retaining ribosome subunits from ES27L Δb1-4 strain, combined with translation factors from wild-type strains, elucidated that iMet retention on the 40S ribosomal subunit causes translation errors. Our study demonstrated the critical role of ES27L in adjusting ribosome association of universally conserved MetAP enzyme to fine-tune iMet processing of key RPs, thereby ensuring the structural integrity and functional accuracy of eukaryotic ribosomes.

DOI: https://doi.org/10.1093/nar/gkaf448
PLoS Genet. 2025 May 27. 21(5): e1011677

The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance.

Joseph K McKenna, Yalan Wu, Praveen Sonkusre, Caleb K Sinclear, Raj Chari, Andres M Lebensohn.

WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

DOI: https://doi.org/10.1371/journal.pgen.1011677
Cell. 2025 May 19. pii: S0092-8674(25)00514-8. [Epub ahead of print]

De novo assembly of nuclear stress bodies rearranges and enhances NFIL3 to restrain acute inflammatory responses.

Xiao-Qi Liu, Pan Li, Bao-Qing Gao, Heng-Le Zhu, Liang-Zhong Yang, Yang Wang, Yu-Yao Zhang, Hao Wu, Yu-Hang Pan, Lin Shan, Hongtao Yu, Li Yang, Ling-Ling Chen.

  The membrane-less nuclear stress bodies (nSBs), with satellite III (SatIII) RNAs as the hallmark, are present in primates upon sensing stresses. We report that SatⅢ DNAs, SatⅢ RNAs, and 30 nSB proteins assemble into well-organized structures shortly after stresses. The activated SatⅢ heterochromatin loci rapidly expand, resulting in reduced spatial distance and enhanced expression of adjacent genes, including the transcription suppressor NFIL3, which is known to dampen proinflammatory cytokine production. Rearranging NFIL3 loci within the nSB territory enhances NFIL3 chromatin accessibility and makes NFIL3 promoters more accessible to transcription factors heat shock transcription factor 1 (HSF1) and bromodomain containing 4 (BRD4), which are also recruited to nSBs upon stresses. Human peripheral blood mononuclear cell (PBMC)-derived macrophages under heat shock plus pathogen-associated molecular pattern treatments exhibit increased SatⅢ and NFIL3 expression, the latter of which suppresses key inflammatory cytokines. Importantly, NFIL3 expression positively correlates with SatⅢ activation in septic patients, a process positively correlated to patient survival, highlighting a role of nSBs in restraining inflammatory responses.

Keywords:  NFIL3; inflammatory response; nuclear bodies; nuclear stress bodies; satellite III; transcription regulation

DOI:  https://doi.org/10.1016/j.cell.2025.05.003
Mol Syst Biol. 2025 May 27.

hu.MAP3.0: atlas of human protein complexes by integration of >25,000 proteomic experiments.

Samantha N Fischer, Erin R Claussen, Savvas Kourtis, Sara Sdelci, Sandra Orchard, Henning Hermjakob, Georg Kustatscher, Kevin Drew.

  Macromolecular protein complexes carry out most cellular functions. Unfortunately, we lack the subunit composition for many human protein complexes. To address this gap we integrated >25,000 mass spectrometry experiments using a machine learning approach to identify >15,000 human protein complexes. We show our map of protein complexes is highly accurate and more comprehensive than previous maps, placing nearly 70% of human proteins into their physical contexts. We globally characterize our complexes using mass spectrometry based protein covariation data (ProteomeHD.2) and identify covarying complexes suggesting common functional associations. hu.MAP3.0 generates testable functional hypotheses for 472 uncharacterized proteins which we support using AlphaFold modeling. Additionally, we use AlphaFold modeling to identify 5871 mutually exclusive proteins in hu.MAP3.0 complexes suggesting complexes serve different functional roles depending on their subunit composition. We identify expression as the primary way cells and organisms relieve the conflict of mutually exclusive subunits. Finally, we import our complexes to EMBL-EBI's Complex Portal ( https://www.ebi.ac.uk/complexportal/home ) and provide complexes through our hu.MAP3.0 web interface ( https://humap3.proteincomplexes.org/ ). We expect our resource to be highly impactful to the broader research community.

Keywords:  Disease Candidates; Machine Learning; Mutually Exclusive; Protein Complex; Protein Interaction

DOI:  https://doi.org/10.1038/s44320-025-00121-5
Nat Commun. 2025 May 27. 16(1): 4909

Restoring calcium crosstalk between ER and mitochondria promotes intestinal stem cell rejuvenation through autophagy in aged Drosophila.

Yao Zhang, Peng Ma, Saifei Wang, Shuxin Chen, Hansong Deng.

Breakdown of calcium network is closely associated with cellular aging. Previously, we found that cytosolic calcium (CytoCa2+) levels were elevated while mitochondrial calcium (MitoCa2+) levels were decreased and associated with metabolic shift in aged intestinal stem cells (ISCs) of Drosophila. How MitoCa2+ was decoupled from the intracellular calcium network and whether the reduction of MitoCa2+ drives ISC aging, however, remains unresolved. Here, we show that genetically restoring MitoCa2+ can reverse ISC functional decline and promote intestinal homeostasis by activating autophagy in aged flies. Further studies indicate that MitoCa2+ and Mitochondria-ER contacts (MERCs) form a positive feedback loop via IP3R to regulate autophagy independent of AMPK. Breakdown of this loop is responsible for MitoCa2+ reduction and ISC dysfunction in aged flies. Our results identify a regulatory module for autophagy initiation involving calcium crosstalk between the ER and mitochondria, providing a strategy to treat aging and age-related diseases.

DOI: https://doi.org/10.1038/s41467-025-60196-4
PLoS Biol. 2025 May 27. 23(5): e3003182

An animal toxin-antidote system kills cells by creating a novel cation channel.

Lews Caro, Aguan D Wei, Christopher A Thomas, Galen Posch, Ahmet Uremis, Michaela C Franzi, Sarah J Abell, Andrew H Laszlo, Jens H Gundlach, Jan-Marino Ramirez, Michael Ailion.

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

DOI: https://doi.org/10.1371/journal.pbio.3003182
Nat Commun. 2025 May 27. 16(1): 4887

Clustering of NLRP3 induced by membrane or protein scaffolds promotes inflammasome assembly.

Elvira Boršić, Taja Železnik Ramuta, Sara Orehek, Mateja Erdani Kreft, Matthias Geyer, Roman Jerala, Iva Hafner-Bratkovič.

NLRP3 is a pattern recognition receptor forming an inflammasome in response to diverse pathogen and self-derived triggers, but molecular insights on NLRP3 activation are still lacking. Here, we drive ectopic NLRP3 to different subcellular locations in NLRP3-deficient macrophages to map the spatial activation profile of NLRP3, and find that NLRP3 variants enriched at the organellar membranes respond to canonical triggers similarly to wild-type NLRP3; however, unlike wild-type, these NLRP3 variants can be activated even in the absence of the polybasic phospholipid-binding segment. Mechanistically, membrane or protein scaffolds mediate NLRP3 clustering, which leads to the unfastening of the inactive NACHT domain conformation preceding the activated NLRP3 oligomer formation. Our data thus suggest that scaffold-promoted clustering is an important step in NLRP3 activation, enabling NLRP3 to sense distinct activator-induced cellular anomalies exhibited via lipid or protein assemblies, thereby establishing NLRP3 as the master sensor of perturbations in cell homeostasis.

DOI: https://doi.org/10.1038/s41467-025-60277-4
Nat Struct Mol Biol. 2025 May 25.

TRIM37 prevents ectopic spindle pole assembly by peptide motif recognition and substrate-dependent oligomerization.

Andrew Bellaart, Amanda Brambila, Jiawei Xu, Francisco Mendez Diaz, Amar Deep, John Anzola, Franz Meitinger, Midori Ohta, Kevin D Corbett, Arshad Desai, Karen Oegema.

Tightly controlled duplication of centrosomes, the primary microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RING-B-box-coiled coil ubiquitin ligase tripartite motif-containing protein 37 (TRIM37), whose loss is associated with elevated chromosome missegregation and the tumor-prone human developmental disorder Mulibrey nanism, prevents the formation of ectopic spindle poles assembling around structured condensates that contain the centrosomal protein centrobin. Here, we show that TRIM37's tumor necrosis factor receptor-associated factor (TRAF) domain, which is unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM family proteins form antiparallel coiled-coil dimers with RING-B-box domains at each end. Oligomerization resulting from RING-RING interactions and conformational regulation through B-box 2-B-box 2 interfaces are essential for TRIM37 to suppress centrobin condensate formation. These results indicate that, similar to antiviral TRIM ligases, TRIM37 activation is coupled to detection of oligomerized substrates, facilitated by recognition of specific motifs in the substrate, to enforce ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

DOI: https://doi.org/10.1038/s41594-025-01562-0
Science. 2025 May 29. eads7373

Predicting expression-altering promoter mutations with deep learning.

Kishore Jaganathan, Nicole Ersaro, Gherman Novakovsky, Yuchuan Wang, Terena James, Jeremy Schwartzentruber, Petko Fiziev, Irfahan Kassam, Fan Cao, Johann Hawe, Henry Cavanagh, Ashley Lim, Grace Png, Jeremy McRae, Abhimanyu Banerjee, Arvind Kumar, Jacob Ulirsch, Yan Zhang, Francois Aguet, Pierrick Wainschtein, Laksshman Sundaram, Adriana Salcedo, Sofia Kyriazopoulou Panagiotopoulou, Delasa Aghamirzaie, Evin Padhi, Ziming Weng, Shan Dong, Damian Smedley, Mark Caulfield, Anne O'Donnell-Luria, Heidi L Rehm, Stephan J Sanders, Anshul Kundaje, Stephen B Montgomery, Mark T Ross, Kyle Kai-How Farh.

Only a minority of patients with rare genetic diseases are currently diagnosed by exome sequencing, suggesting that additional unrecognized pathogenic variants may reside in non-coding sequence. Here, we describe PromoterAI, a deep neural network that accurately identifies non-coding promoter variants which dysregulate gene expression. We show that promoter variants with predicted expression-altering consequences produce outlier expression at both RNA and protein levels in thousands of individuals, and that these variants experience strong negative selection in human populations. We observe that clinically relevant genes in rare disease patients are enriched for such variants and validate their functional impact through reporter assays. Our estimates suggest that promoter variation accounts for 6% of the genetic burden associated with rare diseases.

DOI: https://doi.org/10.1126/science.ads7373
Nat Commun. 2025 May 24. 16(1): 4832

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

Yuichi Furuhata, Gordon Rix, James A Van Deventer, Chang C Liu.

Genetic code expansion (GCE) is a critical approach to the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons (e.g., the amber stop codon). However, evolving new aaRS/tRNA pairs traditionally relies on a labor-intensive process that often yields aaRSs with suboptimal ncAA incorporation efficiencies. Here, we present an OrthoRep-mediated strategy for aaRS evolution, which we demonstrate in 8 independent aaRS evolution campaigns, yielding multiple aaRSs that incorporate an overall range of 13 ncAAs tested. Some evolved systems enable ncAA-dependent translation at single amber codons with similar efficiency as natural translation at sense codons. Additionally, we discover an aaRS that regulated its own expression to enhance ncAA dependency. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms to advance the field of GCE.

DOI: https://doi.org/10.1038/s41467-025-60120-w
EMBO J. 2025 May 28.

Structural insights into lipid membrane binding by human ferlins.

Constantin Cretu, Aleksandar Chernev, Csaba Zoltán Kibédi Szabó, Vladimir Pena, Henning Urlaub, Tobias Moser, Julia Preobraschenski.

  Ferlins are ancient membrane proteins with a unique architecture, and play central roles in crucial processes that involve Ca2+-dependent vesicle fusion. Despite their links to multiple human diseases and numerous functional studies, a mechanistic understanding of how these multi-C2 domain-containing proteins interact with lipid membranes to promote membrane remodelling and fusion is currently lacking. Here we obtain near-complete cryo-electron microscopy structures of human myoferlin and dysferlin in their Ca2+- and lipid-bound states. We show that ferlins adopt compact, ring-like tertiary structures upon membrane binding. The top arch of the ferlin ring, composed of the C2C-C2D region, is rigid and exhibits only little variability across the observed functional states. In contrast, the N-terminal C2B and the C-terminal C2F-C2G domains cycle between alternative conformations and, in response to Ca2+, close the ferlin ring, promoting tight interaction with the target membrane. Probing key domain interfaces validates the observed architecture, and informs a model of how ferlins engage lipid bilayers in a Ca2+-dependent manner. This work reveals the general principles of human ferlin structures and provides a framework for future analyses of ferlin-dependent cellular functions and disease mechanisms.

Keywords:  C2 Domain; Ca2+ Sensing and Signalling; Cryo-EM; Ferlins; Membrane Fusion

DOI:  https://doi.org/10.1038/s44318-025-00463-8
RNA. 2025 May 28. pii: rna.080572.125. [Epub ahead of print]

Tissue-specific SEC31A alternative splicing is regulated by RBM47 and controls lipid transport.

Felix Ostwaldt, Sarah Ploessner, Benjamin Dimos-Roehl, Marco Preussner, Florian Heyd.

  The importance of coat protein complex II (COPII) for protein secretion has been known for decades. However, how large cargo like pro-collagens or chylomicrons are secreted remains incompletely understood, as COPII vesicles are usually too small to accommodate such bulky cargo. Here we introduce alternative splicing as another regulatory layer in controlling secretion of large cargo. We use RNA-Seq data from various human tissues to identify tissues-specific alternative splicing in secretion-associated genes. This identifies an uncharacterized exon in SEC31A, a component of the COPII machinery, whose inclusion is highly tissue-specific, with high inclusion for example in digestive tissues. We show that inclusion of this exon increases lipid transport, thereby connecting SEC31A alternative splicing with the secretion of large cargo. Furthermore, by correlating SEC31A alternative splicing with the expression of RNA-binding proteins across multiple tissues, we identify and then validate RBM47 as the regulator of SEC31A alternative splicing. This serves as proof-of-principle for a broadly applicable in silico approach to facilitate the identification of trans-acting factors controlling tissue-specific alternative splicing.

Keywords:  COPII; SEC31; alternative splicing; lipid transport; secretory pathway

DOI:  https://doi.org/10.1261/rna.080572.125
Nat Struct Mol Biol. 2025 May 25.

Mesoscale regulation of microtubule-organizing centers by the E3 ligase TRIM37.

Zhong Y Yeow, Sonia Sarju, Fang-Chi Chang, Lance Y Xu, Mark van Breugel, Andrew J Holland.

Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well established, less is known about the suppression of noncentrosomal microtubule-organizing centers (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had thus far remained unknown. Here we elucidate the activation process of TRIM37, unveiling a process that initiates with TRAF domain-directed substrate recognition followed by B-box domain-mediated oligomerization and culminates in RING domain dimerization. Using optogenetics, we demonstrate that the E3 activity of TRIM37 is directly coupled to the assembly state of its substrates, being activated only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and echoes the restriction of the human immunodeficiency virus capsid by TRIM5, thus unveiling a conserved activation blueprint among TRIM proteins to control turnover of complexes assembled at the mesoscale level.

DOI: https://doi.org/10.1038/s41594-025-01540-6
Nat Commun. 2025 May 28. 16(1): 4929

RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α.

Hao Liu, Yongqin Tang, Anshu Singh, Joaquim Vong, Julio Cordero, Arthur Mathes, Rui Gao, Yanhan Jia, Boyan K Garvalov, Till Acker, Gernot Poschet, Rüdiger Hell, Marc A Schneider, Joerg Heineke, Thomas Wieland, Guillermo Barreto, Adelheid Cerwenka, Michael Potente, Sofia-Iris Bibli, Rajkumar Savai, Gergana Dobreva.

Defective DNA repair and metabolic rewiring are highly intertwined in promoting the development and progression of cancer. However, the molecular players at their interface remain poorly understood. Here we show that an RNF20-HIF1α axis links the DNA damage response and metabolic reprogramming in lung cancer. We demonstrate that RNF20, which catalyzes monoubiquitylation of histone H2B (H2Bub1), controls Rbx1 expression and thereby the activity of the VHL ubiquitin ligase complex and HIF1α levels. Ablation of a single Rnf20 allele significantly increases the incidence of lung tumors in mice. Mechanistically, Rnf20 haploinsufficiency results in inadequate tumor suppression via the Rnf20-H2Bub1-p53 axis and induces DNA damage, cell growth, epithelial-mesenchymal transition (EMT), and metabolic rewiring through HIF1α-mediated RNA polymerase II promoter-proximal pause release, which is independent of H2Bub1. Importantly, decreased RNF20 levels correlate with increased expression of HIF1α and its target genes, suggesting HIF1α inhibition as a promising therapeutic approach for lung cancer patients with reduced RNF20 activity.

DOI: https://doi.org/10.1038/s41467-025-60223-4
Nat Chem. 2025 May 28.

Structure-informed design of an ultrabright RNA-activated fluorophore.

Mo Yang, Peri R Prestwood, Luiz F M Passalacqua, Sumirtha Balaratnam, Christopher R Fullenkamp, J Winston Arney, Kevin M Weeks, Adrian Ferre-D'Amare, John S Schneekloth.

RNA-based fluorogenic aptamers, such as Mango, are uniquely powerful tools for imaging RNA that activate the fluorescence of a weakly or non-fluorescent small molecule when bound. A central challenge has been to develop brighter, more specific and high-affinity aptamer-ligand systems for cellular imaging. Here we report an ultrabright fluorophore for the Mango II system discovered using a structure-informed, fragment-based small-molecule microarray approach. This dye-termed SALAD1 (structure-informed, array-enabled LigAnD 1)-exhibits subnanomolar aptamer affinity and 3.5-fold brighter fluorescence than Mango II-TO1-biotin pair, a widely used fluorogenic system. Performance was improved by modulating RNA-dye molecular recognition without altering the fluorophore's π-system. High-resolution X-ray structures reveal the binding mode for SALAD1, which exhibits improved pocket occupancy, a more defined binding pose and a unique bonding interaction with potassium. SALAD1 is cell-permeable and facilitates improved in-cell confocal RNA imaging. This work introduces an additional RNA-activated fluorophore demonstrating how fragment-based ligand discovery can be used to create high-performance ligands for RNA targets.

DOI: https://doi.org/10.1038/s41557-025-01832-w