bims-proteo 2025-06-22 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–06–22
forty-one papers selected by
Eric Chevet, INSERM

Accurate de novo design of high-affinity protein-binding macrocycles using deep learning.
System-Wide Profiling of Ubiquitin E3 Ligase Activities with Quantitative Proteomics and Bioinformatics Analysis.
Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma.
Proteasome caspase-like activity regulates stress granules and proteasome condensates.
Proteostasis signatures in human diseases.
Spatiotemporal Regulation of STING Activity by Linear Ubiquitination Governs Antiviral Immunity.
Targeted protein degradation by KLHDC2 ligands identified by high-throughput screening.
USP37 prevents premature disassembly of stressed replisomes by TRAIP.
Investigation of Glutarimide N-Alkylated Derivatives of Lenalidomide.
Integrator complex collapse: A mechanistic route to integrated stress response and disease.
Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62.
Targeted Degradation of Histone Deacetylases via Bypassing E3 Ligase Targeting Chimeras (BYETACs).
Hydroxyurea modulates thiol-disulfide homeostasis in the yeast endoplasmic reticulum.
HADDOCK-Guided modeling and molecular simulations of cereblon-based ternary complexes: Development of novel PROTACs for Ataxia telangiectasia and RAD3-Related (ATR) kinase.
Transgenic expression of endoplasmic reticulum proteins in C. elegans neurons is prone to causing ER stress.
HIV Vpr activates a nucleolar-specific ATR pathway to degrade the nucleolar stress sensor CCDC137.
Regulation of NRF2 by Stably associated Phosphoinositides and Small Heat Shock Proteins in Response to Stress.
DMXL1 promotes recruitment of V1-ATPase to lysosomes upon TRPML1 activation.
The evolutionarily conserved PRP4K-CHMP4B/vps32 splicing circuit regulates autophagy.
Synergizing multiresolution simulations, interface redesign, and hotspot mapping to decipher pathogenic mutation-driven structural modulation in VCP.
Dysfunctional mitochondria trap proteins in the intermembrane space.
Hyperacute response proteins synthesized on γ-tubulin-FTO-MARK4 translation microdomains regulate cancer's acute stress response.
Therapeutic potential of BOLD-100, a GRP78 inhibitor, enhanced by ATR inhibition in pancreatic ductal adenocarcinoma.
Targeted O-GlcNAcylation of CK2α Triggers Its Ubiquitin-Proteasome Degradation and Alters Downstream Phosphorylation.
Lysosomal membrane permeabilization enhances the anticancer effects of POLR1 (RNA polymerase I) transcription inhibitors.
The integrated stress response fine-tunes stem cell fate decisions upon serine deprivation and tissue injury.
The RNA-binding E3 ligase MKRN2 selectively disrupts Il6 translation to restrain inflammation.
Resting Ca2+ fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming.
Protocol for evaluating compound uptake and RNase L co-localization in live cells using fluorescence-based binding, competition assay, and confocal microscopy.
Profiling Ligand-Induced Changes in Nuclear Localization Using Proximity Labeling-Coupled Chemoproteomics.
SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion.
Polyvalent folate receptor-targeting chimeras for degradation of membrane proteins.
Enhancing anti-tumor immunity of natural killer cells through targeting IL-15R signaling.
No evidence that human GIGYF2 interacts with GRB10: implications for human disease.
Perturb-Multimodal: A platform for pooled genetic screens with imaging and sequencing in intact mammalian tissue.
AlphaDesign: a de novo protein design framework based on AlphaFold.
Regulation of redox homeostasis by ATF4-MTHFD2 axis during white adipose tissue browning.
Metabolic control of glycosylation forms for establishing glycan-dependent protein interaction networks.
A stress-sensing circuit signals to the central pacemaker to reprogram circadian rhythms.
A deep generative model for deciphering cellular dynamics and in silico drug discovery in complex diseases.
Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale.

Nat Chem Biol. 2025 Jun 20.

Accurate de novo design of high-affinity protein-binding macrocycles using deep learning.

Stephen A Rettie, David Juergens, Victor Adebomi, Yensi Flores Bueso, Qinqin Zhao, Alexandria N Leveille, Andi Liu, Asim K Bera, Joana A Wilms, Alina Üffing, Alex Kang, Evans Brackenbrough, Mila Lamb, Stacey R Gerben, Analisa Murray, Paul M Levine, Maika Schneider, Vibha Vasireddy, Sergey Ovchinnikov, Oliver H Weiergräber, Dieter Willbold, Joshua A Kritzer, Joseph D Mougous, David Baker, Frank DiMaio, Gaurav Bhardwaj.

Developing macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource intensive and provide little control over binding mode. Despite progress in protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic binders against protein targets of interest. We tested 20 or fewer designed macrocycles against each of four diverse proteins and obtained binders with medium to high affinity against all targets. For one of the targets, Rhombotarget A (RbtA), we designed a high-affinity binder (Kd < 10 nM) despite starting from the predicted target structure. X-ray structures for macrocycle-bound myeloid cell leukemia 1, γ-aminobutyric acid type A receptor-associated protein and RbtA complexes match closely with the computational models, with a Cα root-mean-square deviation < 1.5 Å to the design models. RFpeptides provides a framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

DOI: https://doi.org/10.1038/s41589-025-01929-w
Methods Mol Biol. 2025 ;2921 345-359

System-Wide Profiling of Ubiquitin E3 Ligase Activities with Quantitative Proteomics and Bioinformatics Analysis.

Yue Chen, Yao Gong.

  Ubiquitination is an essential protein posttranslational modification in eukaryotes governing protein homeostasis and ubiquitin-mediated cellular signaling. In human cells, the dynamics and substrate-specificity of ubiquitination are mediated by over 600 genetically encoded E3 ligases. Dysregulation of E3 ligase activities could disrupt protein turnover, gene expression, and signal transduction, which is widely implicated in diseases such as cancer and aging-related diseases. In this report, we presented a computational workflow, UbE3-APA, to profile E3 ligase activities based on quantitative proteomics data and bioinformatics analysis. We validated the strategy with published datasets from the analysis of cells and tissues with genetic knockout or knockdown of specific E3 ligases and demonstrated that the strategy can be applied to both data-dependent or data-independent quantitative proteomics datasets.

Keywords:  Activity-based profiling; E3 ligase; Quantitative proteomics; UbE3-APA; Ubiquitination

DOI:  https://doi.org/10.1007/978-1-0716-4502-4_19
Mol Cell. 2025 Jun 11. pii: S1097-2765(25)00469-1. [Epub ahead of print]

Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma.

Christopher Nardone, Jingjing Gao, Hyuk-Soo Seo, Julian Mintseris, Lucy Ort, Matthew C J Yip, Milen Negasi, Anna K Besschetnova, Nolan Kamitaki, Steven P Gygi, Sirano Dhe-Paganon, Nikhil C Munshi, Mariateresa Fulciniti, Michael E Greenberg, Sichen Shao, Stephen J Elledge, Xin Gu.

  The midnolin-proteasome pathway degrades many nuclear proteins without ubiquitination, but how it operates mechanistically remains unclear. Here, we present structures of the midnolin-proteasome complex, revealing how established proteasomal components are repurposed to enable a unique form of proteolysis. While the proteasomal subunit PSMD2/Rpn1 binds to ubiquitinated or ubiquitin-like (Ubl) proteins, we discover that it also interacts with the midnolin nuclear localization sequence, elucidating how midnolin's activity is confined to the nucleus. Likewise, PSMD14/Rpn11, an enzyme that normally cleaves ubiquitin chains, surprisingly functions non-enzymatically as a receptor for the midnolin Ubl domain, positioning the substrate-binding Catch domain directly above the proteasomal entry site to guide substrates into the proteasome. Moreover, we demonstrate that midnolin downregulation is critical for the survival of myeloma cells by stabilizing the transcription factor substrate IRF4. Our findings uncover the mechanisms underlying the midnolin-proteasome pathway and midnolin downregulation as a driver of multiple myeloma.

Keywords:  IRF4; PSMD14/Rpn11; PSMD2/Rpn1; midnolin; myeloma; proteasome; ubiquitin-independent

DOI:  https://doi.org/10.1016/j.molcel.2025.05.030
Front Cell Dev Biol. 2025 ;13 1570499

Proteasome caspase-like activity regulates stress granules and proteasome condensates.

Shirel Steinberger, Julia Adler, Nadav Myers, Yosef Shaul.

  The 20S proteasome maintains cellular protein homeostasis, particularly during stress responses. In a previous study, we identified numerous 20S proteasome substrates through mass spectrometry analysis of peptides generated from cellular extracts degraded by purified 20S proteasome. Many substrates were found to be components of liquid-phase separation, such as stress granules (SGs). Here, we demonstrate the degradation products arise from the caspase-like (CL) proteasomal activity. To investigate the functional implications of CL activity, we generated cell lines devoid of CL function by introducing the PSMB6 T35A mutation. These mutant cells exhibited slower growth rates, heightened sensitivity to stress, and activation of the unfolded protein response (UPR), as indicated by elevated levels of spliced XBP1 (sXBP1) and stress markers. Cells were subjected to arsenite and osmotic stress to assess their responses. Our findings reveal that CL activity is crucial for efficient SG assembly but does not significantly affect SG clearance. Interestingly, in these mutant cells, proteasomes were more cytoplasmic under normal conditions but formed nuclear condensates/granules (PGs) upon NaCl osmotic stress. However, the PGs were unstable and rapidly dispersed. These findings underscore the important role of the proteasome's CL activity in managing stress-induced dynamics of liquid-liquid phase, highlighting its importance in cellular adaptation to proteotoxic and genotoxic stress conditions.

Keywords:  20S proteasome; intrinsically disordered proteins; proteasome caspase-like activity; proteasome condensates; stress granules

DOI:  https://doi.org/10.3389/fcell.2025.1570499
PLoS Comput Biol. 2025 Jun;21(6): e1013155

Proteostasis signatures in human diseases.

Christine M Lim, Michele Vendruscolo.

The protein homeostasis (proteostasis) network maintains the proteome in a functional state. Although this network has been comprehensively mapped, its perturbations in disease remain incompletely characterised. To address this problem, here we define the proteostasis signatures, which represent the characteristic patterns of change in the proteostasis network associated with disease. We performed a large-scale, pan-disease analysis across 32 human diseases spanning 7 disease types. We first identified unique proteostasis perturbations in specific disease states. We then uncovered distinctive signatures differentiating disease types, pointing to a range of proteostasis mechanisms in disease development. Next, we tracked the temporal evolution of proteostasis signatures, revealing shifts in proteostasis disruption over the course of disease progression. Finally, we demonstrated how smoking, a major risk factor for many diseases, impairs proteostasis in a manner similar to disease, potentially creating a predisposed environment for disease onset. These results illustrate the opportunities offered by the study of human diseases from the perspective of proteostasis signatures.

DOI: https://doi.org/10.1371/journal.pcbi.1013155
Adv Sci (Weinh). 2025 Jun 19. e2417660

Spatiotemporal Regulation of STING Activity by Linear Ubiquitination Governs Antiviral Immunity.

Yong Zhang, Yesheng Fu, Lihua Qiang, Mengyuan Zhao, Zhe Lu, Zhuo Zhao, Guoping Chen, Zehui Lei, Qiyao Chai, Pupu Ge, Bingxi Li, Jing Wang, Cui Hua Liu, Lingqiang Zhang.

  The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene protein (STING) signaling plays a critical role in innate immunity and must be tightly regulated to maintain immune homeostasis, but the mechanism underlying the spatiotemporal regulation of this pathway remains largely elusive. Here, it is shown that during DNA viral infection, the linear ubiquitin chain assembly complex (LUBAC) and ovarian tumor deubiquitinase with linear linkage specificity (OTULIN) reversibly catalyze the linear ubiquitination of STING. At the early stage of the infection, LUBAC promotes STING linear ubiquitination to drive its trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus through binding to the Sec24b subunit of the coat protein complex II (COPII) complex. Later on, OTULIN is recruited to TANK1 binding kinase 1 (TBK1)-phosphorylated STING and removes its linear ubiquitin chains, thus preventing excessive antiviral immune responses. Together, the study uncovers a linear ubiquitination-governed spatiotemporal regulatory mechanism that fine-tunes STING-driven antiviral immunity.

Keywords:  HOIP; OTULIN; STING; antiviral immunity; linear ubiquitination

DOI:  https://doi.org/10.1002/advs.202417660
Elife. 2025 Jun 16. pii: RP106844. [Epub ahead of print]14

Targeted protein degradation by KLHDC2 ligands identified by high-throughput screening.

Han Zhou, Tonglian Zhou, Wenli Yu, Liping Liu, Yeonjin Ko, Kristen A Johnson, Ian A Wilson, Peter G Schultz, Michael J Bollong.

  Proteolysis-targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC-compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high-throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high-throughput screening.

Keywords:  E3 ligase; KLHDC2; PROTAC; biochemistry; chemical biology; high-throughput screening; human

DOI:  https://doi.org/10.7554/eLife.106844
Nat Commun. 2025 Jun 18. 16(1): 5333

USP37 prevents premature disassembly of stressed replisomes by TRAIP.

Olga V Kochenova, Giuseppina D'Alessandro, Domenic Pilger, Ernst Schmid, Sean L Richards, Marcos Rios Garcia, Satpal S Jhujh, Andrea Voigt, Vipul Gupta, Christopher J Carnie, R Alex Wu, Nadia Gueorguieva, Simon Lam, Grant S Stewart, Johannes C Walter, Stephen P Jackson.

The eukaryotic replisome, which consists of the CDC45-MCM2-7-GINS (CMG) helicase, replicative polymerases, and several accessory factors, sometimes encounters proteinaceous obstacles that threaten genome integrity. These obstacles are targeted for removal or proteolysis by the E3 ubiquitin ligase TRAIP, which associates with the replisome. However, TRAIP must be carefully regulated to avoid inappropriate ubiquitylation and disassembly of the replisome. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. Furthermore, TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when converging replisomes stall. Finally, guided by AlphaFold-Multimer, we discovered that binding to CDC45 mediates USP37's response to topological stress. We propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination.

DOI: https://doi.org/10.1038/s41467-025-60139-z
ACS Chem Biol. 2025 Jun 16.

Investigation of Glutarimide N-Alkylated Derivatives of Lenalidomide.

Farah Kabir, Toshiaki Sonobe, Qian Zhu, Nandini Vallavoju, Yuka Amako, Christina M Woo.

Lenalidomide is a thalidomide derivative that engages the E3 ligase substrate receptor cereblon (CRBN) to promote targeted protein degradation. Lenalidomide possesses a glutarimide moiety, which is responsible for CRBN engagement, and an isoindoline moiety, which promotes neosubstrate recruitment. Modification of the glutarimide is a generalizable prodrug strategy to inhibit CRBN binding for the selective activation of CRBN-dependent activity, yet these compounds may possess CRBN-independent effects. We prepared six N-alkylated glutarimide derivatives and found CRBN-independent effects on TNFα inhibition and selective effects in the cell viability profiles. Evaluation of selected compounds by global proteomics in KG1a cells reveals that the downregulation of Rab28 is CRBN-independent and mediated by autophagy. Finally, we developed a representative prodrug to demonstrate the enzymatic release of lenalidomide. Collectively, although some CRBN-independent properties are observed, modification of glutarimide is a generally viable strategy to prevent CRBN engagement in a prodrug strategy.

DOI: https://doi.org/10.1021/acschembio.5c00272
Mol Cell. 2025 Jun 19. pii: S1097-2765(25)00463-0. [Epub ahead of print]85(12): 2268-2270

Integrator complex collapse: A mechanistic route to integrated stress response and disease.

Beatrice Valtorta, Hana Hanzlikova.

In a recent Cell article, Baluapuri et al.1 show that loss of the Integrator (INT) complex activates the integrated stress response via double-stranded RNA from incomplete pre-mRNAs, revealing a link to INT-related neurodevelopmental diseases and potential therapeutic targets.

DOI: https://doi.org/10.1016/j.molcel.2025.05.024
Nat Commun. 2025 Jun 17. 16(1): 5328

Aneuploidy-induced proteostasis disruption impairs mitochondrial functions and mediates aggregation of mitochondrial precursor proteins through SQSTM1/p62.

Prince Saforo Amponsah, Jan-Eric Bökenkamp, Olha Kurpa, Svenja Lenhard, Anna Myronova, Daniel Osmar Vega Velazquez, Celina Hirschelmann, Christian Behrends, Johannes M Herrmann, Markus Räschle, Zuzana Storchová.

Aneuploidy, or aberrant chromosomal content, disrupts cellular proteostasis through altered expression of numerous proteins. Aneuploid cells accumulate SQSTM1/p62-positive cytosolic bodies, exhibit impaired protein folding, and show altered proteasomal and lysosomal activity. Here, we employ p62 proximity- and affinity-based proteomics to elucidate p62 interactors in aneuploid cells and observe an enrichment of mitochondrial proteins. Increased protein aggregation and colocalization of p62 with both novel interactors and mitochondrial proteins is further confirmed by microscopy. Compared to parental diploids, aneuploid cells suffer from mitochondrial defects, including perinuclearly-clustered mitochondrial networks, elevated reactive oxygen species levels, reduced mitochondrial DNA abundance, and impaired protein import, leading to cytosolic accumulation of mitochondrial precursor proteins. Overexpression of heat shock proteins in aneuploid cells mitigates protein aggregation and decreases the colocalization of p62 with the mitochondrial protein TOMM20. Thus, proteotoxic stress caused by chromosome gains results in the sequestration of mitochondrial precursor proteins into cytosolic p62-bodies, thereby compromising mitochondrial function.

DOI: https://doi.org/10.1038/s41467-025-60857-4
ACS Med Chem Lett. 2025 Jun 12. 16(6): 1155-1162

Targeted Degradation of Histone Deacetylases via Bypassing E3 Ligase Targeting Chimeras (BYETACs).

Tao Sun, Shiyang Zhai, Beate König, Irina Honin, Cindy-Esther Kponomaizoun, Finn K Hansen.

  Targeted protein degradation (TPD) through heterobifunctional molecules to initiate ubiquitination and facilitate subsequent degradation has emerged as a powerful therapeutic strategy. Most heterobifunctional molecules designed for TPD function primarily through a limited set of E3 ligases, which restricts this therapeutic approach to specific tissues that express the necessary ligases. Herein, we have developed a novel series of heterobifunctional bypassing E3 targeting chimeras (BYETACs) for the targeted degradation of histone deacetylases (HDACs). To this end, a ubiquitin-specific protease 14 (USP14) inhibitor is utilized for the first time as a novel ligand that can directly bind to the 26S proteasome subunit RPN1. Subsequent conjugation of the USP14 ligand with the HDAC inhibitor vorinostat yielded HDAC BYETACs that effectively and preferentially reduced HDAC1 protein levels in multiple myeloma MM.1S cells.

Keywords:  Bypassing E3 targeting chimeras (BYETACs); cancer; histone deacetylases (HDACs); targeted protein degradation (TPD); ubiquitin-specific protease 14 (USP14)

DOI:  https://doi.org/10.1021/acsmedchemlett.5c00193
Life Sci Alliance. 2025 Aug;pii: e202503225. [Epub ahead of print]8(8):

Hydroxyurea modulates thiol-disulfide homeostasis in the yeast endoplasmic reticulum.

Yuki Takano, Yuki Ishiwata-Kimata, Ryo Ushioda, Yukio Kimata, Kunio Nakatsukasa.

Hydroxyurea (HU) has been extensively used in laboratory settings to induce S-phase arrest and checkpoint activation. Furthermore, it has a history of clinical use as a cost-effective chemotherapeutic agent. Nevertheless, there is still uncertainty regarding its precise pharmacology, side effects, and toxicity, particularly in terms of its impact on organelle homeostasis. Here, we demonstrate that in budding yeast, HU specifically inhibits the endoplasmic reticulum-associated degradation (ERAD) of luminal misfolded proteins (ERAD-L pathway), an effect that is independent of S-phase arrest. In contrast, HU did not affect the degradation of misfolded ER membrane proteins or the degradation of cytosolic proteins. The selective inhibition of ERAD-L by HU is likely attributable to the formation of disulfide bonds in cysteine residues in luminal substrates, which must be reduced before their retrotranslocation to the cytosol. We further demonstrate that HU plays a role in alleviating reductive stress phenotypes observed in cells lacking Ero1, which is essential for oxidative protein folding in the ER. We propose that HU functions as a modulator of thiol-disulfide homeostasis in the ER lumen.

DOI: https://doi.org/10.26508/lsa.202503225
Comput Biol Med. 2025 Aug;pii: S0010-4825(25)00921-7. [Epub ahead of print]194 110570

HADDOCK-Guided modeling and molecular simulations of cereblon-based ternary complexes: Development of novel PROTACs for Ataxia telangiectasia and RAD3-Related (ATR) kinase.

Anne-Christin Sarnow, Husam Nassar, Abdallah M Alfayomy, Dina Robaa, Wolfgang Sippl.

  Proteolysis-targeting chimeras (PROTACs) offer a novel therapeutic strategy for degrading disease-causing proteins, but designing effective degraders remains challenging. PROTACs function by inducing a ternary complex between the target protein and an E3 ligase, requiring structural insights for rational design. Key factors include linker optimization, attachment points, and warhead refinement. Computational approaches, particularly protein-protein docking, are essential for modeling ternary complexes and predicting critical interactions. However, existing docking methods struggle with cereblon (CRBN)-based ternary complexes. To address this, we introduce a computational approach combining HADDOCK protein-protein docking with induced fit PROTAC docking. Validated against 26 crystal structures from the Protein Data Bank (PDB), this method demonstrated high accuracy, especially for CRBN-based complexes. Additionally, molecular dynamics (MD) simulations of CRBN-BRD4-BD1 complexes (PDB IDs 6BN7, 6BOY) provided insights into complex stability through buried surface area and radius of gyration calculations. This validated approach was then applied to five Ataxia telangiectasia and RAD3-related (ATR) kinase PROTACs, enabling modeling in the absence of experimental structures. Our method provides a robust framework for optimizing and designing novel PROTACs targeting diverse proteins.

Keywords:  Ataxia telangiectasia and RAD3-Related (ATR) kinase; Cereblon; HADDOCK; Molecular dynamics simulation; Protein-protein docking; Proteolysis targeting chimera (PROTAC); Ternary complex modeling

DOI:  https://doi.org/10.1016/j.compbiomed.2025.110570
MicroPubl Biol. 2025 ;2025

Transgenic expression of endoplasmic reticulum proteins in C. elegans neurons is prone to causing ER stress.

Junhyun Park, Shaul Yogev.

In studying the endomembrane system, organelle-specific markers tagged with fluorescent proteins are used to visualize individual organelles. However, whether the expression of organelle marker perturbs the organelle's biology is not always apparent. We report that expression of a GFP-tagged Endoplasmic Reticulum (ER) protein causes low levels of ER stress that are challenging to detect in control animals. This stress is revealed only once the ER-associated degradation (ERAD) pathway is compromised. Our results highlight the vulnerability of the ER and suggest that the possible contribution of ER stress to phenotypes obtained with transgenic markers should be considered when interpreting the phenotypes.

DOI: https://doi.org/10.17912/micropub.biology.001547
Nucleic Acids Res. 2025 Jun 06. pii: gkaf531. [Epub ahead of print]53(11):

HIV Vpr activates a nucleolar-specific ATR pathway to degrade the nucleolar stress sensor CCDC137.

Karly A Nisson, Rishi S Patel, Yennifer Delgado, Mehdi Bouhaddou, Lucie Etienne, Oliver I Fregoso.

The lentiviral accessory protein Vpr engages an extensive network of cellular pathways to drive diverse host consequences. Of its many phenotypes, CRL4A-E3 ubiquitin ligase complex co-option, DNA damage response (DDR) engagement, and G2/M arrest are conserved and thus proposed to be functionally important. How Vpr effects these functions and whether they explain how Vpr dysregulates additional cellular pathways remain unclear. Here we leverage the ability of Vpr to deplete the nucleolar protein CCDC137 to understand how Vpr-induced DDR activation impacts nucleolar processes. We characterize CCDC137 as an indirect Vpr target whose degradation does not correlate with Vpr-induced G2/M arrest. Yet, degradation is conserved among Vpr from the pandemic HIV-1 and related SIVcpz/SIVgor, and it is triggered by genomic insults that activate a nucleolar ATR pathway in a manner similar to camptothecin. We determine that Vpr causes ATR-dependent features of nucleolar stress that correlate with CCDC137 degradation, including redistribution of nucleolar proteins, altered nucleolar morphology, and repressed ribosome biogenesis. Together, these data distinguish CCDC137 as a non-canonical Vpr target that may serve as a sensor of nucleolar disruption, and in doing so, identify a novel role for Vpr in nucleolar stress.

DOI: https://doi.org/10.1093/nar/gkaf531
J Biol Chem. 2025 Jun 12. pii: S0021-9258(25)02217-3. [Epub ahead of print] 110367

Regulation of NRF2 by Stably associated Phosphoinositides and Small Heat Shock Proteins in Response to Stress.

Changliang Chen, Noah D Carrillo, Mo Chen, Tianmu Wen, Poorwa Awasthi, Richard A Anderson, Vincent L Cryns.

  Reactive oxygen species (ROS) are generated by aerobic metabolism, and their deleterious effects are buffered by the cellular antioxidant response, which prevents oxidative stress. The nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of the antioxidant response. Basal levels of NRF2 are kept low by ubiquitin-dependent degradation of NRF2 by E3 ligases, including the Kelch-like ECH-associated protein 1 (KEAP1). Here, we show that the stability and function of NRF2 is regulated by the type I phosphatidylinositol phosphate kinase γ (PIPKIγ), which binds NRF2 and is required to stably couple phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to NRF2 in response to oxidative stress. Stress also induces the interaction of the small heat shock protein HSP27 and NRF2 and this interaction is enhanced by PtdIns(4,5)P2. Silencing PIPKIγ or HSP27 destabilizes NRF2, reduces expression of its target gene HO-1, and sensitizes cells to oxidative stress. These data demonstrate an unexpected collaboration between phosphoinositides, which are stably coupled to NRF2, and HSP27, which is recruited to NRF2 by a phosphoinositide-dependent mechanism to regulate NRF2 stability and function. These findings also point to PIPKIγ and HSP27 as drug targets to destabilize NRF2 in cancer.

Keywords:  NRF2; PtdIns(4,5)P(2); nucleus; phosphoinositide; small heat shock proteins: oxidative stress

DOI:  https://doi.org/10.1016/j.jbc.2025.110367
Nat Struct Mol Biol. 2025 Jun 17.

DMXL1 promotes recruitment of V1-ATPase to lysosomes upon TRPML1 activation.

Chan Lee, Matthew J G Eldridge, Miguel A Gonzalez-Lozano, Thomas Bresnahan, Zachary Niday, Donato Del Camino, Tao Fu, Joao A Paulo, Magdalene M Moran, Sophie Helaine, J Wade Harper.

Lysosomes, central hydrolytic organelles, are regulated by ion flow, including calcium and protons, via transporters and channels to maintain an acidified lumen for hydrolytic activity. TRPML1, a lysosomal ion channel, effluxes cations upon activation, promoting rapid conjugation of ATG8 proteins to the lysosomal membrane in a process known as conjugation of ATG8 to single membranes (CASM). However, our understanding of how TRPML1 activation reorganizes the lysosomal proteome is poorly understood. Here, we identify DMXL1 as a key regulator of lysosomal homeostasis through quantitative proteomics of lysosomes during TRPML1 activation by the agonist MLSA5. DMXL1 is recruited to lysosomes and Salmonella-containing vacuoles, both in a CASM-dependent manner. As the mammalian ortholog of yeast Rav1, DMXL1 assembles with Rav2 ortholog ROGDI and WDR7, and associates with V0 and V1 subunits of the lysosomal V-ATPase. TRPML1 activation drives V1 subunit recruitment to lysosomes in a DMXL1- and DMXL2-dependent manner. DMXL1- and DMXL2-deficient cells display reduced V1-ATPase recruitment, increased lysosomal pH and diminished hydrolytic capacity. Using AlphaFold modeling supported by cross-linking proteomics, we identify interaction interfaces within the DMXL1-ROGDI-WDR7 complex, as well as an ATP6V1A binding interface in DMXL1, whose mutation affects interaction and function. Our findings suggest CASM-dependent DMXL1 recruitment, coupled with V-ATPase assembly, is critical for maintaining lumenal pH and lysosomal function in response to TRPML1 activation.

DOI: https://doi.org/10.1038/s41594-025-01581-x
Cell Rep. 2025 Jun 17. pii: S2211-1247(25)00641-2. [Epub ahead of print]44(7): 115870

The evolutionarily conserved PRP4K-CHMP4B/vps32 splicing circuit regulates autophagy.

Sabateeshan Mathavarajah, Sandhya Chipurupalli, Elias B Habib, William D Kim, Megan M Aoki, Dale P Corkery, Kennedy I T Whelan, Jordan Lukacs, Melis Erkan, Victor D Martinez, Kevin S Smith, Stephen B Montgomery, Jayme Salsman, Robert J Huber, Graham Dellaire.

  The pre-mRNA processing factor 4 kinase (PRP4K) is an essential gene in animal cells, making interrogation of its function challenging. Here, we report characterization of a viable knockout model of PRP4K in the social amoeba Dictyostelium discoideum, revealing a function for PRP4K in splicing events controlling autophagy. When prp4k knockout amoebae undergo multicellular development, we observe defects in differentiation linked to abnormal autophagy and aberrant secretion of stalk cell inducer c-di-GMP. Autophagosome-lysosome fusion is impaired after PRP4K loss in both human cell lines and amoebae. PRP4K loss results in mis-splicing and reduced expression of the ESCRT-III gene CHMP4B in human cells and its ortholog vps32 in Dictyostelium, and re-expression of CHMP4B or Vps32 cDNA (respectively) restores normal autophagosome-lysosome fusion in PRP4K-deficient cells. Thus, our work reveals a PRP4K-CHMP4B/vps32 splicing circuit regulating autophagy that is conserved over at least 600 million years of evolution.

Keywords:  CHMP4B; CP: Cell biology; CP: Developmental biology; Dictyostelium; ESCRT III; PRP4K; PRPF4B; autophagy; splicing kinase

DOI:  https://doi.org/10.1016/j.celrep.2025.115870
Comput Biol Med. 2025 Aug;pii: S0010-4825(25)00911-4. [Epub ahead of print]194 110560

Synergizing multiresolution simulations, interface redesign, and hotspot mapping to decipher pathogenic mutation-driven structural modulation in VCP.

Amar Jeet Yadav, Aditya K Padhi.

  Valosin-containing protein (VCP/p97), a pivotal AAA+ ATPase, orchestrates proteostasis via ER-associated degradation (ERAD), ubiquitin-mediated proteolysis, and organelle surveillance. Pathogenic missense mutations, notably Arg95Gly (R95G) within the evolutionarily conserved double-ψ β-barrel (DPBB) of its N-terminal domain, are implicated in proteinopathies including IBMPFD and ALS. To decode the structural-dynamics perturbations underpinning R95G-driven dysfunction, we integrated AlphaFold3-based modeling, protein-peptide docking, and multiscale enhanced-sampling molecular dynamics (MD) simulations-spanning 1.2 μs all-atom, 12 μs coarse-grained, and umbrella sampling regimes. Our findings reveal that R95G disrupts the β-barrel integrity, destabilizes long-range domain coupling, and engenders conformational heterogeneity deleterious to gp78 cofactor recruitment. Free-energy landscapes of the mutant highlight enthalpically disfavored, low-occupancy binding conformers, corroborated by MM/PBSA-based end-state binding free energy and potential of mean force (PMF) analyses, which indicate impaired binding thermodynamics. Interface hotspot mapping pinpoints dynamic perturbations at critical residues that propagate allosteric decoupling and morphological distortion of the binding interface. Collectively, our results delineate a mechanistic cascade-from local β-barrel destabilization to global interaction network disruption-underlying VCP's functional impairment in disease states. This work provides a computationally derived structural framework to inform targeted biophysical validation and the rational design of therapeutic strategies aimed at rescuing VCP function in IBMPFD and ALS.

Keywords:  Double-psi β-barrel; Interface redesign; Molecular docking; Molecular dynamics simulation; Umbrella sampling; Valosin-containing protein

DOI:  https://doi.org/10.1016/j.compbiomed.2025.110560
EMBO J. 2025 Jun 16.

Dysfunctional mitochondria trap proteins in the intermembrane space.

Tamara Flohr, Markus Räschle, Johannes M Herrmann.

  The accumulation of mitochondrial precursor proteins in the cytosol due to mitochondrial dysfunction compromises cellular proteostasis and is a hallmark of diseases. Why non-imported precursors are toxic and how eukaryotic cells prevent their accumulation in the cytosol is still poorly understood. Using a proximity labeling-based assay to globally monitor the intramitochondrial location of proteins, we show that, upon mitochondrial dysfunction, many mitochondrial matrix proteins are sequestered in the intermembrane space (IMS); something we refer to as "mitochondrial triage of precursor proteins" (MitoTraP). MitoTraP is not simply the result of a general translocation block at the level of the inner membrane, but specifically directs a subgroup of matrix proteins into the IMS, many of which are constituents of the mitochondrial ribosome. Using the mitoribosomal protein Mrp17 (bS6m) as a model, we found that IMS sequestration prevents its mistargeting to the nucleus, potentially averting interference with assembly of cytosolic ribosomes. Thus, MitoTraP represents a novel, so far unknown mechanism of the eukaryotic quality control system that protects the cellular proteome against the toxic effects of non-imported mitochondrial precursor proteins.

Keywords:  Intermembrane Space; Mitochondria; Nucleolus; Protein Targeting; Ribosome

DOI:  https://doi.org/10.1038/s44318-025-00486-1
Cell Rep. 2025 Jun 18. pii: S2211-1247(25)00642-4. [Epub ahead of print]44(7): 115871

Hyperacute response proteins synthesized on γ-tubulin-FTO-MARK4 translation microdomains regulate cancer's acute stress response.

Bruno Saleme, Saymon Tejay, Kléouforo-Paul Dembélé, Rabih Abou Farraj, Yongneng Zhang, Yongsheng Liu, Aristeidis E Boukouris, Sotirios D Zervopoulos, Alois Haromy, Yuan-Yuan Zhao, Shelly Braun, William Saleme, Xuejun Sun, Richard P Fahlman, Mark Glover, Adam Kinnaird, Gopinath Sutendra, Evangelos D Michelakis.

  Compared to normal cells, cancer cells are particularly resistant to stress, and their immediate response to stress is critical for subsequent adaptation, a major clinical challenge. With unbiased proteomics and transcriptomics, we identify a list of hyperacute response proteins (HARPs) translated from pre-existing mRNAs within 20 min of diverse stresses in several cancer cells, despite the known suppressed global translation in stress. HARP mRNAs are translated on microtubule-associated translation microdomains (MATMs) located on γ-tubulin, which host FTO and specialized distinct cytoskeletal ribosomes. FTO exits the nucleus immediately after stress and is activated by microtubule-associated kinase MARK4, demethylating a translation-inhibiting m6A mRNA methylation signature and facilitating compartmentalized HARP translation on MATMs, while non-HARP mRNAs remain inhibited. FTO or MARK4 inhibition suppresses HARP synthesis and increases apoptosis after various stresses, including chemotherapy. γ-tubulin, FTO, and MARK4 are therapeutic targets, as they comprehensively promote HARP translation, a potential Achilles' heel for cancer's resistance to physiologic or therapeutic stress.

Keywords:  CP: Cancer; CP: Molecular biology; cancer resistance; m6A; microtubules; ribosomes; stress response; translation; γ-tubulin

DOI:  https://doi.org/10.1016/j.celrep.2025.115871
Cell Commun Signal. 2025 Jun 13. 23(1): 281

Therapeutic potential of BOLD-100, a GRP78 inhibitor, enhanced by ATR inhibition in pancreatic ductal adenocarcinoma.

Su In Lee, Ah-Rong Nam, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Yoojin Jeong, Sea Young Choo, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh.

  Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and resistance to conventional therapies, necessitating novel treatments. The high proliferative rate and protein synthesis in PDAC induce endoplasmic reticulum (ER) stress, with Glucose-Regulated Protein 78 (GRP78), a key regulator of ER stress and the Unfolded Protein Response (UPR), playing a pivotal role in PDAC progression. Despite its relevance, GRP78-targeted therapies remain unexplored in PDAC. BOLD-100, a novel GRP78 inhibitor, presents a potential therapeutic approach by disrupting GRP78 transcription, though its effects on PDAC have yet to be fully elucidated. Here, we found that BOLD-100 induces PDAC cell death through the UPR pathway activation, leading to CHOP-dependent apoptosis. BOLD-100 generates reactive oxygen species (ROS), inducing R-loop formation that triggers a DNA damage response via the ATR/Chk1 axis. BOLD-100 synergizes with AZD6738, an ATR inhibitor, to enhance anti-tumor efficacy compared to either agent alone in both in vitro and in vivo models. These findings suggest that BOLD-100, especially in combination with an ATR inhibitor, represents a promising therapeutic option for patients with PDAC.

Keywords:  ATR; CHOP; DNA damage response; ER stress; GRP78; Pancreatic cancer; ROS

DOI:  https://doi.org/10.1186/s12964-025-02242-8
ACS Chem Biol. 2025 Jun 16.

Targeted O-GlcNAcylation of CK2α Triggers Its Ubiquitin-Proteasome Degradation and Alters Downstream Phosphorylation.

Tongyang Xu, Bowen Ma, Yuanpei Li, Zhihao Guo, Miaomiao Zhang, Billy Wai-Lung Ng.

O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is an important post-translational modification (PTM), yet dissecting its protein-specific functions has remained challenging. Here, we applied our previously reported chemical biology tool, the O-GlcNAcylation Targeting Chimera (OGTAC), to specifically induce O-GlcNAcylation of the casein kinase II subunit α (CK2α) at Ser347 in living cells. We found that this targeted O-GlcNAcylation destabilized CK2α through ubiquitin-proteasome degradation and enhanced its interaction with cereblon (CRBN). Overexpression and knockdown experiments also indicated CK2α as a substrate of the Cullin-RING E3 ubiquitin ligase 4-CRBN (CRL4CRBN) E3 ligase complex. Furthermore, the OGTAC-induced O-GlcNAcylation of CK2α reprogrammed phosphorylation of Akt and PFKP. These findings reveal that a single O-GlcNAc modification can serve as a molecular switch, controlling the protein stability and downstream phosphorylation of CK2α. More broadly, our results highlight the profound utility of the OGTAC-mediated O-GlcNAcylation to interrogate its cellular functions with specificity, overcoming limitations inherent to prior global perturbation methods.

DOI: https://doi.org/10.1021/acschembio.5c00223
Autophagy. 2025 Jun 18. 1-20

Lysosomal membrane permeabilization enhances the anticancer effects of POLR1 (RNA polymerase I) transcription inhibitors.

Lucille Ferret, Jonathan G Pol, Allan Sauvat, Gautier Stoll, Karla Alvarez-Valadez, Alexandra Muller, Julie Le Naour, Felix Peyre, Gerasimos Anagnostopoulos, Isabelle Martins, Maria Chiara Maiuri, Harald Wodrich, Lionel Guittat, Jean-Louis Mergny, Guido Kroemer, Mojgan Djavaheri-Mergny.

  Lysosomes contribute to the development of drug resistance through various mechanisms that include drug sequestration and the activation of adaptive stress pathways. While inhibitors of DNA-to-RNA transcription exhibit potent anticancer effects, the role of lysosomes in modulating responses to such transcription inhibitors remains largely unexplored. This study investigates this aspect in the context of two potent POLR1 (RNA polymerase I) transcription inhibitors, CX-3543 (quarfloxin) and CX-5461 (pidnarulex). Unexpectedly, CX-3543 was found to accumulate within lysosomes, leading to lysosomal membrane permeabilization (LMP) and the subsequent activation of cellular stress adaptation pathways, including those regulated by the transcription factor TFEB and autophagy. Disrupting TFEB or autophagy increased cell sensitivity to CX-3543, highlighting the cytoprotective role of these processes in counteracting CX-3543-induced cell death. Moreover, targeting lysosomal membranes with chloroquine derivatives or blue light exposure induced substantial LMP, releasing compound CX-3543 from lysosomes. This effect enhanced both the inhibition of DNA-to-RNA transcription and CX-3543-induced cell death. Similar effects were observed when chloroquine derivatives were combined with CX-5461. Additionally, combining CX-3543 with the chloroquine derivative DC661 more effectively reduced the fibrosarcoma growth in immunocompetent mice than either agent alone. Altogether, our results reveal an unanticipated lysosome-related mechanism that contributes to cancer cell resistance to POLR1 inhibitors and propose a strategy to overcome this resistance.Abbreviations: ATG7: autophagy related 7; ATG13: autophagy related 13; Baf A1: bafilomycin A1; CTSB: cathepsin B; DKO: double knockout; G4: Guanine quadruplex; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LGALS3: galectin 3; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; NCL: nucleolin; POLR1: RNA polymerase I; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TFE3: transcription factor E3; ULK1: unc-51 like autophagy activating kinase 1.

Keywords:  Autophagy; TFEB; cancer; cell death; guanine quadruplex ligands; resistance to therapy

DOI:  https://doi.org/10.1080/15548627.2025.2497614
Cell Metab. 2025 Jun 12. pii: S1550-4131(25)00266-9. [Epub ahead of print]

The integrated stress response fine-tunes stem cell fate decisions upon serine deprivation and tissue injury.

Jesse S S Novak, Lisa Polak, Sanjeethan C Baksh, Douglas W Barrows, Marina Schernthanner, Benjamin T Jackson, Elizabeth A N Thompson, Anita Gola, M Deniz Abdusselamoglu, Alain R Bonny, Kevin A U Gonzales, Julia S Brunner, Anna E Bridgeman, Katie S Stewart, Lynette Hidalgo, June Dela Cruz-Racelis, Ji-Dung Luo, Shiri Gur-Cohen, H Amalia Pasolli, Thomas S Carroll, Lydia W S Finley, Elaine Fuchs.

  Epidermal stem cells produce the skin's barrier that excludes pathogens and prevents dehydration. Hair follicle stem cells (HFSCs) are dedicated to bursts of hair regeneration, but upon injury, they can also reconstruct, and thereafter maintain, the overlying epidermis. How HFSCs balance these fate choices to restore physiologic function to damaged tissue remains poorly understood. Here, we uncover serine as an unconventional, non-essential amino acid that impacts this process. When dietary serine dips, endogenous biosynthesis in HFSCs fails to meet demands (and vice versa), slowing hair cycle entry. Serine deprivation also alters wound repair, further delaying hair regeneration while accelerating re-epithelialization kinetics. Mechanistically, we show that HFSCs sense each fitness challenge by triggering the integrated stress response, which acts as a rheostat of epidermal-HF identity. As stress levels rise, skin barrier restoration kinetics accelerate while hair growth is delayed. Our findings offer potential for dietary and pharmacological intervention to accelerate wound healing.

Keywords:  dietary intervention; epidermal stem cells; fate selection; hair follicle stem cells; hair regrowth; integrated stress response; serine metabolism; tissue regeneration; tissue repair; wound healing

DOI:  https://doi.org/10.1016/j.cmet.2025.05.010
Nat Immunol. 2025 Jun 16.

The RNA-binding E3 ligase MKRN2 selectively disrupts Il6 translation to restrain inflammation.

Zhou Yu, Xuelian Li, Jiaying Huang, Jueyu Pan, Jiale Cheng, Ping Liu, Mingjin Yang, Taoyong Chen, Qian Zhang, Yumei Zhou, Jiacheng Wu, Taotao Han, Jingnan Li, Yue Xu, Mingyue Wen, Xuan Zhang, Chunmei Wang, Xuetao Cao.

E3 ligases and RNA-binding protein-mediated dysregulation of proinflammatory cytokines leads to autoimmune and inflammatory diseases. However, whether RNA-binding E3 ligases can regulate specific proinflammatory cytokine expression remains unclear. Here we found that the RNA-binding E3 ligase MKRN2 selectively inhibits the expression of interleukin-6 (IL-6) in lipopolysaccharide-activated macrophages. LysM-Cre+Mkrn2fl/fl mice showed increased amounts of IL-6 in the serum after lipopolysaccharide treatment and exhibited increased severity of experimental colitis, which was associated with increased IL-6. Expression of MKRN2 negatively correlated with expression of IL-6 in clinical samples from individuals with ulcerative colitis and rheumatoid arthritis. Mechanistically, after binding to Il6 messenger RNA, MKRN2 linked K29 polyubiquitin chains to the Lys 179 residue of PAIP1, a translation initiation coactivator, which blocked PAIP1-eIF4A interaction and thus inhibited the translational efficiency of Il6 mRNA. Our findings provide mechanistic insight and potential therapeutic strategies for inflammatory autoimmune diseases by disrupting translation of specific proinflammatory cytokines.

DOI: https://doi.org/10.1038/s41590-025-02183-x
Cell Mol Life Sci. 2025 Jun 14. 82(1): 238

Resting Ca2+ fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming.

Caroline Fecher, Annemarie Sodmann, Felicitas Schlott, Juliane Jaepel, Franziska Schmitt, Isabella Lengfelder, Thorsten Bischler, Bernhard Nieswandt, Konstanze F Winklhofer, Robert Blum.

  Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+ fluxes. The data showed that endoplasmic reticulum (ER) Ca²⁺ depletion, induced by sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition, resulted to prolonged Ca²⁺ influx and mitochondrial fragmentation within 10 to 30 min. This mitochondrial fragmentation could be prevented in Ca2+-free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate-early cell stress and transcriptional reprogramming.

Keywords:  Calcium signaling; ER calcium; ER calcium leak; Mitochondrial fragmentation; Resting calcium fluxes; Store-operated calcium entry; Transcriptome changes

DOI:  https://doi.org/10.1007/s00018-025-05745-2
STAR Protoc. 2025 Jun 14. pii: S2666-1667(25)00310-7. [Epub ahead of print]6(3): 103904

Protocol for evaluating compound uptake and RNase L co-localization in live cells using fluorescence-based binding, competition assay, and confocal microscopy.

Elias Khaskia, Raphael I Benhamou.

  Using ribonuclease targeting chimera (RIBOTAC) technology, fluorescent probes enable real-time visualization of RNase L localization and interaction dynamics. Here, we present a protocol to assess probe uptake, binding specificity, and RNase L co-localization in live cells using a fluorescent-based binding and competition assay combined with confocal microscopy. We provide step-by-step instructions for live-cell imaging and quantitative fluorescence analysis, enabling researchers to monitor RNA degradation pathways and evaluate the effects of RNA-targeting small molecules with high spatial resolution. For complete details on the use and execution of this protocol, please refer to Khaskia et al.1.

Keywords:  Cell Biology; Microscopy; Molecular Biology; Molecular/Chemical Probes

DOI:  https://doi.org/10.1016/j.xpro.2025.103904
Methods Mol Biol. 2025 ;2921 73-91

Profiling Ligand-Induced Changes in Nuclear Localization Using Proximity Labeling-Coupled Chemoproteomics.

Qianni Peng, Eranthie Weerapana.

  The nuclear proteome encompasses diverse proteins that regulate critical cellular functions, including histone modifications, chromatin structure, and transcription. Mutations to many of these nuclear proteins correlate with the onset of diseases such as cancer. Due to the disease relevance of nuclear proteins, drug development efforts have focused on identifying small-molecule modulators of nuclear protein function. Covalent ligands provide a promising strategy to therapeutically target nuclear proteins that lack distinct substrate binding pockets. In particular, chemoproteomic strategies have enabled the identification of ligandable sites within the proteome, with a particular emphasis on covalent targeting of cysteine residues. Nuclear proteins are typically poorly represented in chemoproteomic workflows that utilize whole-cell lysates due to the low abundance of these proteins and the localization of nuclear proteins in multiple cellular compartments. To specifically focus on the nuclear proteome, we coupled proximity labeling using a histone-TurboID construct with chemoproteomics. Notably, this platform can be utilized to identify ligandable sites within the nuclear proteome, and monitor changes in nuclear localization and chromatin association upon exposure to covalent ligands. Here, we describe the steps required to generate histone-TurboID expressing cell lines, and apply tandem mass tag (TMT)-based quantitative proteomics to monitor protein localization changes induced by covalent ligands. Together, this methodology provides a streamlined approach toward identifying covalent ligands that regulate nuclear protein function.

Keywords:  Chemoproteomics; Chromatin association; Nuclear cysteine ligandability; Nuclear localization; Proximity labeling; TurboID

DOI:  https://doi.org/10.1007/978-1-0716-4502-4_4
PLoS Pathog. 2025 Jun 20. 21(6): e1013295

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion.

Jianchao Zhang, Andrew Kennedy, Daniel Macedo de Melo Jorge, Lijuan Xing, Whitney Reid, Sarah Bui, Joseph Joppich, Molly Rose, Sevval Ercan, Qiyi Tang, David Ginsburg, Andrew W Tai, Yanzhuang Wang.

The COVID-19 pandemic is caused by the enveloped virus SARS-CoV-2. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Blocking endoplasmic reticulum (ER) to Golgi trafficking dramatically inhibits SARS-CoV-2 assembly and secretion without reducing viral genome replication. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion without affecting viral entry, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to modulate viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

DOI: https://doi.org/10.1371/journal.ppat.1013295
Nat Chem Biol. 2025 Jun 13.

Polyvalent folate receptor-targeting chimeras for degradation of membrane proteins.

Dian Xiao, Jingwen Dong, Fei Xie, Xun Feng, Jianfeng Wang, Xin Xu, Borui Tang, Cuicui Sun, Yuting Wang, Wu Zhong, Hongbin Deng, Xinbo Zhou, Song Li.

Lysosome-targeting chimeras (LYTACs) represent a revolutionary targeted protein degradation technology. However, the advancement of LYTACs faces substantial challenges due to the limited diversity of lysosome-trafficking receptors. In this study, we identified folate receptor α (FRα) as a new class of lysosome-trafficking receptors capable of facilitating the degradation of membrane proteins. Leveraging a polyvalent crosslinking strategy, we developed FRα-targeting chimeras (FRTACs), including enhanced green fluorescent protein-targeting FR-Ctx and PD-L1-targeting FR-Atz. The optimized FRTACs demonstrated subnanomolar potency in eliminating cell-surface targets, with efficacy dependent on both FRα expression and lysosomal activity. Specifically, FR-Ctx inhibited cancer cell proliferation, while FR-Atz enhanced T cell-mediated cytotoxicity against tumor cells. FR-Atz exhibited robust PD-L1 degradation efficiency in vivo and elicited tumor-specific immune responses by reprogramming the tumor microenvironment from an immunosuppressive to an immunostimulatory state in both RM-1 and humanized B16F10 mouse models. These findings establish FRTACs as a promising platform for the design of tumor-targeting LYTACs.

DOI: https://doi.org/10.1038/s41589-025-01924-1
Cancer Cell. 2025 Jun 10. pii: S1535-6108(25)00220-X. [Epub ahead of print]

Enhancing anti-tumor immunity of natural killer cells through targeting IL-15R signaling.

Iva Nikolic, Joseph Cursons, Benjamin Shields, Stephane Chappaz, Harrison Sudholz, Xiangpeng Meng, Patrick Constantinescu, Reshma Vijayakumaran, Michael D'Angelo, Momeneh Foroutan, David Ladd, Matthew Veldman, Jason Glab, Tahlia Procter, Hae-Young Park, Julian Contet, Felix Deuss, Kahlia Wong, Yi Sun, Richard Berry, Jai Rautela, Nicholas D Huntington.

  Interleukin-15 receptor (IL-15R) agonists induce anti-tumor immunity in pre-clinical models. However, dose-limiting toxicity has hampered their clinical development. We performed genome-wide CRISPR screens to reveal the complete IL-15R signaling mechanism in natural killer (NK) cells and discovered that ubiquitin-dependent IL-15R degradation is the dominant mechanism restraining IL-15R signaling. Key hits included the NEDD8 E2-conjugating enzyme UBE2F, the ubiquitin E3-ligase ARIH2, and Cullin-5 RING E3 ligase (CRL5) members. We found that UBE2F was required for neddylation and activation of CUL5, whereas ARIH2 contributed to CRL5-mediated IL-15RB degradation. Ablation of ARIH2 or UBE2F increased IL-15RB surface expression and enhanced signaling, resulting in proinflammatory cytokine production and augmented natural and CAR-mediated cytotoxicity. In mice lacking Arih2, Rnf7, or Ube2f, we observed that the IL-15R hyperresponsive NK cells exhibited superior in vivo anti-tumor immunity against primary and disseminated metastatic tumors. Thus, we have identified the enzymes UBE2F and ARIH2 as tractable immunotherapy drug targets.

Keywords:  CRISPR screening; cytokine receptors; cytokine signaling; interleukin-15; natural killer cells; post-translational modifications

DOI:  https://doi.org/10.1016/j.ccell.2025.05.011
Life Sci Alliance. 2025 Sep;pii: e202503334. [Epub ahead of print]8(9):

No evidence that human GIGYF2 interacts with GRB10: implications for human disease.

Jung-Hyun Choi, Israel Shpilman, Niaz Mahmood, Shaghayegh Farhangmehr, Ulrich Braunschweig, Nathalia Gomide Cruz, Jun Luo, Reese Jalal Ladak, Angelos Pistofidis, Jiannis Ragoussis, T Martin Schmeing, Seyed Mehdi Jafarnejad, Benjamin J Blencowe, Nahum Sonenberg.

GIGYF2 (growth factor receptor-bound protein 10 [GRB10]-interacting GYF [glycine-tyrosine-phenylalanine] protein 2) reduces mRNA stability and translation via microRNAs, ribosome quality control, and several RNA-binding proteins. GIGYF2 was first identified in mouse cell lines as an interacting partner with GRB10, which binds to the insulin receptor and the insulin-like growth factor receptor 1. Mutations in the human GIGYF2 gene were reported in autism. In mouse models, Gigyf2 mutations engender several diseases. It was therefore thought that the GIGYF2-associated disease in humans is caused by defective GRB10 signaling. We show here that GIGYF2 does not interact with GRB10 in human cell lines, as determined by co-immunoprecipitation and proximity ligation assays. The lack of interaction is explained by the absence of the critical GYF domain-binding PPGΦ sequence in the human GRB10 protein. These results contrast with the current understanding that a GIGYF2/GRB10 complex is associated with human disease via insulin receptor and insulin-like growth factor receptor 1 signaling and underscore alternative mechanisms responsible for the observed phenotypes associated with mutations in the human GIGYF2 gene.

DOI: https://doi.org/10.26508/lsa.202503334
Cell. 2025 Jun 11. pii: S0092-8674(25)00572-0. [Epub ahead of print]

Perturb-Multimodal: A platform for pooled genetic screens with imaging and sequencing in intact mammalian tissue.

Reuben A Saunders, William E Allen, Xingjie Pan, Jaspreet Sandhu, Jiaqi Lu, Thomas K Lau, Karina Smolyar, Zuri A Sullivan, Catherine Dulac, Jonathan S Weissman, Xiaowei Zhuang.

  Metazoan life requires the coordinated activities of thousands of genes in spatially organized cell types. Understanding the basis of tissue function requires approaches to dissect the genetic control of diverse cellular and tissue phenotypes in vivo. Here, we present Perturb-Multimodal (Perturb-Multi), a paired imaging and sequencing method to construct large-scale, multimodal genotype-phenotype maps in tissues with pooled genetic perturbations. Using imaging, we identify perturbations in individual cells while simultaneously measuring their gene expression profiles and subcellular morphology. Using single-cell sequencing, we measure full transcriptomic responses to the same perturbations. We apply Perturb-Multi to study hundreds of genetic perturbations in the mouse liver. Our data suggest the genetic regulators and mechanisms underlying the dynamic control of hepatocyte zonation, the unfolded protein response, and steatosis. Perturb-Multi accelerates discoveries of the genetic basis of complex cell and tissue physiology and provides critical training data for emerging machine learning models of cellular function.

Keywords:  RCA-MERFISH; hepatocyte stress response; in vivo pooled screening; lipid droplet accumulation; liver zonation; machine learning morphology; multimodal phenotyping; multiplexed RNA imaging; multiplexed protein imaging; scRNA-seq

DOI:  https://doi.org/10.1016/j.cell.2025.05.022
Mol Syst Biol. 2025 Jun 17.

AlphaDesign: a de novo protein design framework based on AlphaFold.

Michael A Jendrusch, Alessio L J Yang, Elisabetta Cacace, Jacob Bobonis, Carlos G P Voogdt, Sarah Kaspar, Kristian Schweimer, Cecilia Perez-Borrajero, Karine Lapouge, Jacob Scheurich, Kim Remans, Janosch Hennig, Athanasios Typas, Jan O Korbel, S Kashif Sadiq.

  De novo protein design is of fundamental interest to synthetic biology, with a plethora of computational methods of various degrees of generality developed in recent years. Here, we introduce AlphaDesign, a hallucination-based computational framework for de novo protein design developed with maximum generality and usability in mind, which combines AlphaFold with autoregressive diffusion models to enable rapid generation and computational validation of proteins with controllable interactions, conformations and oligomeric state without the requirement for class-dependent model re-training or fine-tuning. We apply our framework to design and systematically validate in vivo active inhibitors of a family of bacterial phage defense systems with toxic effectors called retrons, paving the way towards efficient, rational design of novel proteins as biologics.

Keywords:  AlphaFold; Anti-phage Defense Proteins; Computational Biology; De Novo Protein Design; Machine Learning

DOI:  https://doi.org/10.1038/s44320-025-00119-z
Redox Biol. 2025 Jun 09. pii: S2213-2317(25)00228-9. [Epub ahead of print]85 103715

Regulation of redox homeostasis by ATF4-MTHFD2 axis during white adipose tissue browning.

Rehna Paula Ginting, Hoang-Anh Pham-Bui, Choijamts Munkhzul, Siti Aisyah Fuad, Ahyeon Son, Jong-Seok Moon, Jaeseok Han, Mihye Lee, Min-Woo Lee.

Maintaining redox balance is crucial for mitochondrial homeostasis. During browning of white adipocytes, both the quality and quantity of mitochondria undergo dramatic changes. However, the mechanisms controlling the redox balance in the mitochondria during this process remain unclear. In this study, we demonstrate that thermogenic activation occurs before mitochondrial biogenesis during cold-induced browning of inguinal white adipose tissue (iWAT) and is accompanied by increased mitochondrial stress and integrated stress response (ISR) signaling. Specifically, cold exposure enhances the expression of ATF4, an ISR effector. Adipocyte-specific deletion of ATF4 results in increased energy expenditure, but paradoxically leads to a lower core body temperature, and heightened pro-inflammation in iWAT after cold exposure, which is restored by the antioxidant, MitoQ. Mechanistically, ATF4 regulates the redox balance through MTHFD2, an enzyme involved in mitochondrial redox homeostasis by NADPH generation. Cold exposure upregulates MTHFD2 expression in an ATF4-dependent manner, and its inhibition by DS18561882 in vivo leads to impaired cold-induced mitochondrial respiration similar to the effects of ATF4 loss. These findings suggest that ATF4 is essential for redox balance via MTHFD2, thereby affecting tissue homeostasis during iWAT browning.

DOI: https://doi.org/10.1016/j.redox.2025.103715
Proc Natl Acad Sci U S A. 2025 Jun 24. 122(25): e2422936122

Metabolic control of glycosylation forms for establishing glycan-dependent protein interaction networks.

Xingyu Liu, Li Yi, Zongtao Lin, Siyu Chen, Shunyang Wang, Ying Sheng, Carlito B Lebrilla, Benjamin A Garcia, Yixuan Xie.

  Protein-protein interactions (PPIs) are crucial for comprehending the molecular mechanisms and signaling pathways underlying diverse biological processes and disease progression. However, investigating PPIs involving membrane proteins is challenging due to the complexity and heterogeneity of glycosylation. To tackle this challenge, we developed an approach termed glycan-dependent affinity purification coupled with mass spectrometry (GAP-MS), specifically designed to characterize changes in glycoprotein PPIs under varying glycosylation conditions. GAP-MS integrates metabolic control of glycan profiles in cultured cells using small molecules referred to as glycan modifiers with affinity purification followed by mass spectrometry analysis (AP-MS). Here, GAP-MS was applied to characterize and compare the interaction networks under five different glycosylation states for four bait glycoproteins: BSG, CD44, EGFR, and SLC3A2. This analysis identified a network comprising 156 interactions, of which 131 were determined to be glycan dependent. Notably, the GAP-MS analysis of BSG provided distinct information regarding glycosylation-influenced interactions compared to the commonly used glycosylation site mutagenesis approach combined with AP-MS, emphasizing the unique advantages of GAP-MS. Collectively, GAP-MS presents distinct insights over existing methods in elucidating how specific glycosylation forms impact glycoprotein interactions. Additionally, the glycan-dependent interaction networks generated for these four glycoproteins serve as a valuable resource for guiding future functional investigations and therapeutic developments targeting the glycoproteins discussed in this study.

Keywords:  AP–MS; glycoprotein; glycosylation; interactome; mass spectrometry

DOI:  https://doi.org/10.1073/pnas.2422936122
Sci Adv. 2025 Jun 20. 11(25): eadr7960

A stress-sensing circuit signals to the central pacemaker to reprogram circadian rhythms.

Maria E Yurgel, Claire Gao, John J O'Malley, Qijun Tang, Noam Yanay, Alison R Bashford, Jesse J Zhan, Andrew Lutas, Michael J Krashes, Haiqing Zhao, Mario A Penzo, Samer Hattar.

The circadian system provides a temporal framework for animals to anticipate environmental events, including threats. However, the effects of stressors on the circadian system remain poorly understood. Here, we demonstrate that, in mice, stressors shift the phase of the central pacemaker, housed in the suprachiasmatic nucleus (SCN), through glutamatergic inputs from the anterior paraventricular nucleus of the thalamus (aPVT). Unlike light, which can phase delay or advance the central pacemaker, stressors consistently induce delays, effects attenuated by inhibiting aPVT neurons. Stressors robustly activate AVP-expressing neurons within the SCN and are associated with inhibition of VIP-expressing neurons, whereas light strongly activates VIP-expressing neurons with minimal effects on AVP-expressing neurons. Pairing stressors with light reveals distinct time-dependent interactions, enhancing phase delays at early night but abolishing phase advances at late night. Our findings uncover distinct SCN microcircuits that differentially encode light and stressors, providing insights into how environmental cues modulate circadian timing.

DOI: https://doi.org/10.1126/sciadv.adr7960
Nat Biomed Eng. 2025 Jun 20.

A deep generative model for deciphering cellular dynamics and in silico drug discovery in complex diseases.

Yumin Zheng, Jonas C Schupp, Taylor Adams, Geremy Clair, Aurelien Justet, Farida Ahangari, Xiting Yan, Paul Hansen, Marianne Carlon, Emanuela Cortesi, Marie Vermant, Robin Vos, Laurens J De Sadeleer, Ivan O Rosas, Ricardo Pineda, John Sembrat, Melanie Königshoff, John E McDonough, Bart M Vanaudenaerde, Wim A Wuyts, Naftali Kaminski, Jun Ding.

Human diseases are characterized by intricate cellular dynamics. Single-cell transcriptomics provides critical insights, yet a persistent gap remains in computational tools for detailed disease progression analysis and targeted in silico drug interventions. Here we introduce UNAGI, a deep generative neural network tailored to analyse time-series single-cell transcriptomic data. This tool captures the complex cellular dynamics underlying disease progression, enhancing drug perturbation modelling and screening. When applied to a dataset from patients with idiopathic pulmonary fibrosis, UNAGI learns disease-informed cell embeddings that sharpen our understanding of disease progression, leading to the identification of potential therapeutic drug candidates. Validation using proteomics reveals the accuracy of UNAGI's cellular dynamics analysis, and the use of the fibrotic cocktail-treated human precision-cut lung slices confirms UNAGI's predictions that nifedipine, an antihypertensive drug, may have anti-fibrotic effects on human tissues. UNAGI's versatility extends to other diseases, including COVID, demonstrating adaptability and confirming its broader applicability in decoding complex cellular dynamics beyond idiopathic pulmonary fibrosis, amplifying its use in the quest for therapeutic solutions across diverse pathological landscapes.

DOI: https://doi.org/10.1038/s41551-025-01423-7
Cell Rep Methods. 2025 Jun 13. pii: S2667-2375(25)00120-1. [Epub ahead of print] 101084

Combination of in vivo and in vitro phosphoproteomics determines the PP2A target repertoire on proteome scale.

Melanie Brunner, Zehan Hu, Heidy Elkhaligy, Gloria Lampo, Carole Roubaty, Christine Vionnet, Devanarayanan Siva Sankar, Sean J McIlwain, Stéphanie Kaeser-Pebernard, Yongna Xing, Jörn Dengjel.

  Dynamics of protein phosphorylation are regulated by the interplay of kinases and phosphatases. Current mass spectrometry-based phosphoproteomic approaches are extremely powerful in identifying and quantifying tens of thousands of phosphosites in single biological samples. However, whereas the mapping of phosphosites is successfully automated supporting high sample throughput, the characterization of responsible kinases and phosphatases still largely depends on laborious protein biochemical assays. To show direct (de)phosphorylation events, in vitro kinase or phosphatase assays using single substrates or peptide arrays are often used. Here, we describe the development of an in vitro phosphatase assay using whole proteome under native conditions as input. We employ this approach to study the PP1 and PP2A target repertoire, characterizing thousands of potential target sites. Focusing on PPP2R5E/B56ε-containing complexes, we combine in vitro with in vivo phosphoproteomics to characterize bona fide target sites, which highlight the role of PP2A in regulating stress granule assembly.

Keywords:  CAPRIN1; CP: biotechnology; PP1; PP2A; PPP2R5E; kinase; mass spectrometry; phosphatase; proteomics; signaling; stress granule

DOI:  https://doi.org/10.1016/j.crmeth.2025.101084